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Abstract
Aim/hypothesis. Previous studies have shown that pro-
longed glucose infusion causes insulin resistance and
triglyceride accumulation in rat skeletal muscle. In
this study, we investigated a possible relationship be-
tween insulin resistance and the composition of differ-
ent accumulated lipid fractions in rat skeletal muscle.
Methods. Continuous glucose infusion was carried out
in rats for 7 days. Lipids were extracted from skeletal
muscle, separated by thin layer chromatography and
fatty acid composition of phospholipids, triglycerides,
diglycerides, free fatty acids and cholesterol esters
fractions was analysed by gas chromatography. ∆9-
Desaturase mRNA was measured by real time poly-
merase chain reaction. The enzyme activity was mea-
sured in the microsomal fractions.
Results. Prolonged glucose infusion (5 days) increased
the relative content of palmitoleic acid (16:1 N7) sever-
al-fold (2.3- to 5.8-fold) in four out of five lipid frac-
tions and enhanced oleic acid (18:1 N9) two-fold in
three lipid fractions suggesting increased ∆9-desaturase
activity while the content of several polyunsaturated
fatty acids was reduced. In parallel, ∆9-Desaturase
mRNA contents and enzyme activities in skeletal mus-
cle were increased 10-fold, 75-fold, 2.6-fold and 7.7-
fold after 2 and 5 days of glucose infusion, respectively.
Conclusion/interpretation. Our results suggest that
long-term glucose oversupply induces a rapid increase
in ∆9-desaturase expression and enzyme activity in
skeletal muscle which leads to fast and specific chang-
es in fatty acid metabolism possibly contributing to
the insulin resistance in this animal model.
[Diabetologia (2003) 46:203–212]
Keywords Hyperglycaemia, hyperinsulinaemia, insu-
lin resistance, lipid fractions, fatty acid composition,
stearoyl-CoA desaturase.
Received: 2 August 2002 / Revised: 28 October 2002
Published online: 31 January 2003
© Springer-Verlag 2003
Corresponding author: E. D. Schleicher, Department of Endo-
crinology, Metabolism and Pathobiochemistry, Eberhard-Karls-
University Tübingen, Otfried-Mueller-Str. 10, 72076 Tübingen,
Germany
E-mail: enschlei@med.uni-tuebingen.de
Abbreviations: PL, phospholipids; DG, diglycerides; TG, tri-
glycerides; CE, cholesterol ester; GR, glucose-infused rats;
C, control rats; PKC, protein kinase C; SFA, saturated fatty acids;
PUFA, sum of all polyunsaturated fatty acids; MUFA, monoun-
saturated fatty acids; GUFA, all groups of unsaturated fatty acids;
SCD, stearoyl-CoA desaturase; GC. gas chromatography.
Diabetologia (2003) 46:203–212
DOI 10.1007/s00125-002-1015-2
Glucose oversupply increases ∆9-desaturase expression
and its metabolites in rat skeletal muscle
B. Houdali
1
, H. G. Wahl
1
, M. Kresi
1
, V. Nguyen
2
, M. Haap
1
, F. Machicao
1
, H. P. T. Ammon
2
, W. Renn
1
,
E. D. Schleicher
1
, H.-U. Häring
1
1
Department of Endocrinology, Metabolism and Pathobiochemistry, Eberhard-Karls-University Tübingen, Tübingen, Germany
2
Department of Pharmacology, Institute for Pharmaceutical Science, Eberhard-Karls-University, Tübingen, Germany
increased adipocyte mass and impaired insulin regula-
tion of lipolysis [11] could increase NEFA, flux to
other tissues like skeletal muscle thus increasing their
triglyceride storage [12, 13], altering the hepatic glu-
cose output [14] and insulin secretion. Increased fat
accumulation can also act in a paracrine and/or endo-
crine way to promote insulin resistance by thus far un-
known mechanisms [15]. In this context composition
of the fat could also be important since the increase in
saturated FA [4, 5] was associated with insulin resis-
tance. An increase in monounsaturated FAs (MUFA)
in serum FA, in kidney and heart phospholipids frac-
tions of obese Zucker rats in comparison to lean litter-
mates has been reported [16]. In humans, MUFA con-
centrations in muscle phospholipids were positively
correlated with fasting plasma insulin concentrations
but negatively with muscle content of polyunsaturated
Increased intramyocellular fat accumulation coincides
with insulin resistance in humans [1, 2, 3, 4, 5, 6, 7]
and in rodents, [8, 9, 10]. It has been suggested that
fatty acid (PUFA) [4]. The altered FA composition in
lipid membranes of skeletal muscle in insulin-resis-
tant, obese or diabetic rodents and humans could be
caused by an altered FA synthesis pattern. This might
be due to altered enzyme activities catalysing the
elongation and desaturation process of FA [17] in the
liver with subsequent transport to the skeletal muscle
and/or due to local change in FA synthesis pattern, al-
though the rates of de novo lipogenesis are believed to
be low in skeletal muscle in both man and experimen-
tal animals [18].
For the formation of long-chain MUFA and PUFA,
FA are desaturated and elongated with the help of
∆5-, ∆6- and ∆9-desaturases which insert a double
bond at the fifth, sixth and ninth carbon from the
carboxyl terminal, respectively [19, 20]. Elongation
is processed by the ubiquitous elongase, which
inserts two carbon units at the carboxyl terminal of
FA [17].
Numerous reports have focused on the role of
high fat diets on insulin action in skeletal muscle
[21, 22, 23, 24, 25]. Consistently, it has been report-
ed that high fat diet increases the proportion of satu-
rated fatty acids in the skeletal muscle membranes.
Studies in rats with high sucrose diets have shown
that the substitution of carbohydrate for fat could
also result in increased rat muscle triglyceride con-
tent, impaired glucose tolerance [9, 26, 27] and in-
creased long chain fatty acid-CoA accumulation in
skeletal muscle [8]. It seems that high sucrose diets
might cause insulin resistance only when they result
in a positive energy balance leading to more weight
gain than in control animals [27]. Although most of
these studies indicate that dietary carbohydrates in-
fluence fatty acid composition in skeletal muscle and
could lead to insulin resistance, the effect of carbo-
hydrate oversupply on the lipid metabolism of the
different lipid fractions has not been studied explicit-
ly.
The aim of this study was to investigate the rela-
tionship between carbohydrate oversupply, insulin re-
sistance and FA composition in different lipid frac-
tions of skeletal muscle in an animal model as was
originally introduced by another study [28].
Materials and methods
Materials. The materials used in this study were purchased as
follows (company in brackets): Ketamine (Ketanest, Parke-
Davis, Freiburg, Germany), Rompun (Bayer, Leverkusen, Ger-
many) silicone rubber (Silastic, Dow Corning, Midland, Mich.,
USA), heparin (Liquemine, Roche, Grenzach, Switzerland),
swivel (ZAK-Medizintechnik, Munich, Germany), syringe
pump (Perfusor, B. Braun, Melsungen, Germany), 50% glu-
cose (Fresenius, Bad Homburg, Germany), Dismembrator S
(B. Braun, Melsungen, Germany), [9,10-
3
H]stearoyl-CoA
(Biotrend Chemicals, Cologne, Germany), Norit A (Norit,
Düsseldorf, Germany).
Animals. All procedures carried out in this study were ap-
proved by the local Animal Experimentation Ethics Committee
and the “principles of laboratory animal care” (NIH publica-
tion no. 85–23, revised 1985) were followed. Female Wistar
rats weighing about 300 g were purchased from Charles River,
Sulzfeld, Germany and were kept at 22°C with a 12 h light to
darkness cycle and a relative humidity of 55 to 60% during the
whole experimental period. The rats were given free access to
water and standard chow pellet diet (Altromin 1324, Altromin-
Futterwerk, Lage, Germany).
Prolonged glucose infusion into conscious rats. Glucose infu-
sion was done as previously described [29]. After placing the
catheter rats, were allowed to recover for 48 h, after which glu-
cose infusion (2.77 mol/l glucose) was started at a rate of
2 ml/h (GR) compared with 77 mmol/l saline infusion at
2 ml/h (C). Rats were allowed water and chow pellet freely. To
measure plasma glucose and insulin concentrations blood sam-
ples were taken from the tail vein [29]. After day 2 or day 5 of
continuous infusion rats were killed and mixed hind-limb mus-
cles were dissected free of connective tissue and fat, cut into
small pieces and subsequently stored at −80°C and subsequent-
ly analysed for glycogen and triglycerides [30].
Skeletal muscle lipid analysis. Samples of 10 mg skeletal mus-
cle free of visible fat and connective tissue were suspended in
1 ml phosphate-buffered saline, vortexed and further homogeni-
sed by ultrasonication. Subsequently lipids were extracted using
2.5 ml of isopropanol:n-heptane:phosphoric acid (40:20:1, vol/
vol), vortexed and left to stand for 10 min at room temperature.
After centrifugation the supernatant was quantitatively aspirat-
ed and completely dried under nitrogen stream. Extracts were
resolved in 75 µl chloroform:methanol (2:1) and fractionated
by thin-layer chromatography using thin layer plates (Merck,
Darmstadt, Germany) coated with 0.25 mm silica gel. Plates
were preconditioned by heating at 100° C for 2 h and were
developed (20–30 min) using hexan:diethylether:acetic acid
(27:7:1 vol/vol). The plates were allowed to dry on air and the
separated standard lipid fractions were sprayed with 0.5% 2,7-
dichlorofluoresceine in methanol and were visualised under ul-
traviolet light according to the standards. Five lipid fractions,
ie. phospholipids (PL), triglycerides (TG), diglycerides (DG),
NEFA and cholesterol ester (CE) were scraped off and lipids
were extracted with 2 ml methanol:toluol (1:5). Acetylchlorid
(200 µl) was added and agitated for 1 h at 100°C, then cooled
extracts were treated with 5 ml 0.43 M K
2
CO3, mixed for
2 min and centrifuged at 4000 rpm for 10 min. The upper phase
was transferred quantitatively to GC-vials and dried down to
80 µl under a nitrogen stream. Fatty acid methyl esters of the
separated fractions were analysed by gas-liquid chromatogra-
phy using an HP 5890 A apparatus (Hewlett Packard, Wald-
brunn, Germany) equipped with 60 m×0.25 mm i.d. fused silica
column coated with a 0.2 µm film of Rtx 2331 (Restek, Bad
Homburg, Germany) and detected by flame ionisation detector.
Enzyme activity index. Enzyme activity indices were obtained
by relating the amount of the specific substrate to the corre-
sponding product of the respective enzyme [4, 20].
Preparation of microsomal fractions and assay of
∆
9-desatu-
rase activity. The assay was carried out according to a pub-
lished procedure [31]. Aliquots of excised muscles were
weighed (ca. 200 mg) and ground in a liquid nitrogen-cooled
porcelain. Muscles were placed in a liquid nitrogen-cooled
Dismembrator S and powdered at a setting of 2000 rpm for
1 min. Powdered muscles were suspended in 1 ml ice-cold
buffer containing 10 mmol/l Tris, pH 7.4, 1 mmol/l dithiothre-
204 B. Houdali et al.: Glucose oversupply increases ∆9-desaturase expression and its metabolites in rat skeletal muscle
itol and 0.25 mol/l sucrose. Further homogenization was done
with a motor-driven Potter-Elvehjem Teflon-glass tissue grind-
er at a setting 1500 rpm and for approximately ten cycles.
Crude muscle homogenate was then spun at 15 000 g for
20 min. The supernatant was spun in an ultracentrifuge at
100 000 g for 1 h at 4°C using Optima Max centrifuge
(Beckman, Munich, Germany). The supernatant was discarded,
and the microsomal fraction was resuspended in 200 µl of
0.1 mol/l sodium phosphate buffer, pH 7.4 and all steps were
carried out at 4°C. The protein concentration was then mea-
sured using the dye-based Bradford assay (Bio-Rad-Kit) and
∆9-desaturase activity was measured in the microsomal frac-
tion (100 µl) by the generation of
3
H
2
O from the substrate
[9,10-
3
H]stearoyl-CoA [Biotrend Chemicals, Cologne, Germany,
specific activity: 2.2×10
12
TBq /mmol]. Samples were incubat-
ed at 37°C for 5 min, terminated by the addition of 1.3 ml of
ethanol, and spun at 15 000 g for 5 min. Residual substrate was
removed by the addition of 40 mg of Norit A, followed by cen-
trifugation as before, and produced
3
H
2
O was measured in the
supernatant by liquid scintillation counting.
Preparation of total RNA from rat skeletal muscle and
RT-PCR. Isolation of RNA from skeletal muscle, reverse tran-
scription and PCR were carried out [30]. Primer design was
made from a gene sequence of ∆9-desaturase, obtained from
the Genome-GenBank. β-Globin was used as external standard
for quantification and control rats were set at 1 then a compari-
son was made to the corresponding treated rats (x-fold of con-
trols). PCR product sizes were verified by gel electrophoresis
on 2% agarose.
Statistical analysis. All data are expressed as means ± SEM.
Data were analysed using analysis of variance with repeated
measure design. Data on fatty acids composition are expressed
in percent of the corresponding fraction. A p value of less than
0.05 was considered to be statistically significant.
Results
Metabolic effects of continuous glucose infusion. Con-
tinuous glucose infusion into rats for 7 days induced
transient hyperglycaemia and persistent hyperinsulina-
emia. Hyperglycaemia peaked after 24 h of glucose
infusion (Fig. 1A), and then fell continuously reaching
normal values after 5 days (day 5: 7.3±0.4 GR vs
6.9±0.06 mmol/l C) and remained normal after 7 days
despite further continuous glucose infusion. Serum
insulin concentrations followed a similar pattern
(Fig. 1B) but hyperinsulinaemia persisted in GR
throughout the glucose infusion period (day 5:219±
10*** GR vs 38±1 µU/ml C). Saline infusion affected
neither plasma glucose nor insulin concentration in
control rats. Glucosuria was detected in day 2 of the
infusion period and a faster weight gain and lower
food consumption was seen in glucose-infused rats
(Table 1). However, the daily amount of infused
glucose was much more than the decreased food
consumption. Of note, the hyperglycaemic/hyper-
insulinaemic state in day 2 changed to a normo-
glycaemic/hyperinsulinaemic state in day 5. This re-
sulted in a 15-fold increase in muscle glycogen con-
tent after 2 days, which fell to a 3.5-fold increase at
day 5 of glucose infusion, whereas triglyceride con-
tent remained high.
Fatty acid composition in the phospholipid fraction of
skeletal muscle. Prolonged glucose infusion did not
affect the composition of saturated fatty acids, where-
as MUFA increased after day 5 of glucose infusion.
Particularly, palmitoleic acid (16:1 N7) increased
2.4-fold and six-fold compared to control values after
2 and 5 days of glucose infusion, respectively. No
changes were seen in the composition of PUFAs with
B. Houdali et al.: Glucose oversupply increases ∆9-desaturase expression and its metabolites in rat skeletal muscle 205
Fig. 1 (A, B). Time-dependent effect of glucose infusion on
plasma glucose (A) and plasma insulin (B) concentrations in
rats. Continuous glucose infusion (2.77 mol/l) into rats (full
circles) was carried out. Control rats received continuous infu-
sion of (77 mmol/l) NaCl (open circles) at the same infusion
rate of 2 ml/h. Blood samples were taken from the tail vein and
glucose and immunoreactive insulin (IRI) concentrations were
measured. Data represent results of five independent experi-
ments; means ± SEM; *p <0.05, ***p<0.001
the exception of the ω3 fatty acid α-linolenic acid
(18:3 N3) which was reduced in skeletal muscle pho-
spholipids by 50% after 5 days of glucose infusion
(Table 2).
To assess changes in enzyme activities of FA
metabolism indices relating substrate and product of
the respective enzyme reactions were calculated. The
index of ∆9-desaturase (measure of activity) was
increased two-fold and six-fold after day 2 and day 5
of glucose infusion, respectively, whereas no changes
were seen in the activities of ∆5- and ∆6-desaturases
nor in that of elongase.
206 B. Houdali et al.: Glucose oversupply increases ∆9-desaturase expression and its metabolites in rat skeletal muscle
Table 1. Effects of continuous glucose infusion on body
weight, glucosuria and food consumption in rats and on glyco-
gen and triglyceride content of rat skeletal muscle. Rats (n=5)
were continuously infused with 2 ml/h glucose solution
(2.77 mol/l) for 2 or 5 days (GR). Control rats received 2 ml/h
of 77 mmol/l NaCl (C). Data represent means ± SEM; *p<0.05,
***p<0.001
Infusion period Day 2 Day 5
CGRCGR
Glucose infused (mmol/d) 0 133 0 133
Urine glucose Negative Positive Negative Negative
Food consumption (g/d) 10±0.3 7.8±0.2* 12±0.3 7±0.3*
gained weight (g/d) 1.8±0.1 3±0* 1.8±0.1 3.4±0.2*
Glycogen content (mg/g muscle) 3.3±0.7 49±5.6*** 4.5±0.9 15.9±2*
Triglyceride content (µmol/g muscle) 244.9±36 417±42* 166.3±29 426±74*
Table 2. Effects of continuous glucose infusion on fatty acid
composition of phospholipid fraction in rat skeletal muscle.
Rats (n=5) were continuously infused with 2 ml/h glucose so-
lution (2.77 mol/l) for 2 or 5 days (GR). Control rats received
2 ml/h of 77 mmol/l NaCl (C). Lipids were extracted from
skeletal muscle and separated by TLC. The fatty acid composi-
tion of the different lipid was analysed by gas chromatography.
Phospholipid fraction is set at 100% and individual FA are
presented in percent of total phospholipid fraction (means
± SEM); * p<0.05. Important changes are indicated in bold
Glucose Infusion day 2 day 5
Phospholipids Composition (%) Composition (%)
Fatty acids
CGRCGR
14:0 1.37±0.23 1.25±0.36 1.59±0.46 1.20±0.12
15:0 0.75±0.14 0.71±0.27 0.75±0.14 0.58±0.10
16:0 33.6±1.2 32.9±1.09 34.4±0.83 34.1±0.99
18:0 16.3±1.1 16.3±0.47 16.9±0.92 15.7±0.87
20:0 0.19±0.03 0.15±0.00 0.17±0.03 0.16±0.03
22:0 0.26±0.06 0.15±0.01 0.16±0.03 0.22±0.07
∑∑
SFA 52.6±1.08 51.4±1.52 54.0±2.22 51.9±1.26
16:1 N7 0.60±0.09 1.44±0.05* 0.33±0.03 1.99±0.28*
18:1 N9 5.26±0.61 4.50±0.22 4.24±0.31 7.08±0.85*
18:1 N7 1.99±0.09 1.99±0.18 2.03±0.16 2.40±0.10
∑∑
MUFA 7.86±0.60 7.93±0.41 6.59±0.26 11.46±1.11*
18:2 N6 10.38±0.7 10.53±0.91 10.71±0.3 10.53±0.90
18:3 N6 0.06±0.00 0.06±0.00 0.06±0.01 0.07±0.00
20:3 N6 0.34±0.03 0.36±0.02 0.34±0.03 0.38±0.03
20:4 N6 11.96±0.7 12.29±0.87 12.47±0.95 11.57±0.58
22:4 N6 0.12±0.13 0.12±0.05 0.15±0.04 0.15±0.01
∑∑
N6 PUFA 23.31±1.27 25.11±0.61 24.14±1.2 23.06±1.14
18:3 N3 0.34±0.05 0.27±0.02* 0.37±0.02 0.19±0.02*
20:5 N3 0.17±0.03 0.12±0.05 0.15±0.04 0.15±0.01
22:5 N3 1.03±0.10 1.09±0.12 1.17±0.11 1.00±0.08
22:6 N3 14.40±1.22 13.71±1.65 13.20±1.25 11.78±0.58
∑∑
N3 PUFA 15.77±1.26 15.07±1.73 14.74±1.3 12.97±0.66
GUFA 39.58±1.5 40.66±1.59 39.36±2.4 36.57±1.77
N6/N3=PUFA6/3 1.52±0.18 1.52±0.27 1.65±0.08 1.77±0.05
(18:0/16:0)x100 (elongase) 49±4 41±9 49±2 46±3
16:1 N7/16:0 x100 (∆9-desaturase) 2±0.3 4±0.1* 1±0.1 6±1*
(20:4 N6/20:3 N6) (∆ 5 desaturase) 35±2 34±2 37±3 31±3
20:3 N6/18:2 N6 (∆6 desaturase) 33±1 35±3 32±3 37±2
The index of ∆9-desaturase was increased 2.4-fold
whereas that of elongase decreased by about 50%. No
significant changes were seen in ∆5- and ∆6-desatu-
rase activity (Table 3).
Fatty acid composition in the diglycerides fraction.
With the exception of stearic acid (18:0) and arachi-
donic acid (20:0), which were decreased in the muscle
of GR at day 5, prolonged glucose infusion did neither
affect the composition nor the absolute content of sat-
urated fatty acids, whereas MUFAs composition in-
creased (~1.3-fold) after day 2 (GR 20.75±1.16 vs
C 15.9±0.92) and after day 5 (~1.9-fold) (GR 22.46±
1.4 vs C 11.88±1.6) of glucose infusion when com-
pared to control rats. The increased composition of
MUFA was again due to an increased relative content
of palmitoleic acid (16:1 N7) after day 2 (1.9-fold)
(GR 5.03±0.33 vs C 2.67±0.37) and day 5 (4.1-fold)
(GR 6.6±0.67 vs C 1.6±0.67) of glucose infusion. No
changes were seen in the composition of PUFAs.
The index of ∆9-desaturase was increased 2.1-fold
after day 2 (GR 15±2 vs C 7±1) and 4.3-fold after day
5 of glucose infusion (GR 17±2 vs C 4±2). No chang-
es were seen in the activities of ∆5- and ∆6-desatu-
rases, except for ∆5-desaturase after day 5 (1.6-fold
increase) (GR 11±1 vs C 7±1), while elongase activity
index decreased by 25% after day 5 of glucose infu-
sion (GR 45±2 vs C 60±3).
Fatty acid composition in the free fatty acid fraction.
Prolonged glucose infusion did neither affect the com-
position nor the absolute content of saturated fatty
acids, whereas MUFAs composition increased after
day 2 (1.3-fold) (GR 16.87±2.46 vs C 12.7±1.2) and
after day 5 (1.6-fold) of glucose infusion when com-
pared to control rats (GR 18.3±1.1 vs C 11.34±0.88).
Again, the increased composition of MUFA was es-
sentially due to increased palmitoleic acid (16:1 N7)
content (2.5-fold after day 2 and 2.5-fold after day 5)
(2 day: GR 5±0.3 vs C 2±0.4; 5 day: GR 5±0.6 vs
C 2±0.4) whereas no changes were seen for PUFAs.
Fatty acid composition in the triglycerides fraction.
With exception of stearic acid (18:0), which was rela-
tively decreased in GR muscle after day 5, prolonged
glucose infusion did neither affect the composition nor
B. Houdali et al.: Glucose oversupply increases ∆9-desaturase expression and its metabolites in rat skeletal muscle 207
Table 3. Effects of continuous glucose infusion on fatty acid
composition of triglycerides fraction. Rats (n=5) were continu-
ously infused with 2 ml/h glucose solution (2.77 mol/l) and
fatty acid content in the lipid fraction was analysed. Triglyce-
ride fraction is set at 100% and individual FA are shown in
percent of total triglyceride fraction (means ± SEM). Important
changes in fatty acids in the treated group compared with the
control group are marked with * (p<0.05) and indicated in bold
Glucose Infusion day 2 day 5
Triglycerides Composition (%) Composition (%)
Fatty acids
CGRCGR
14:0 2.52±0.12 2.51±0.45 2.82±0.85 2.10±0.42
15:0 0.34±0.07 0.68±0.29 0.59±0.17 0.32±0.03
16:0 31.74±1.1 33.03±1.4 31.15±2.6 33.7±0.8
18:0 5.5±0.13 5.4±0.32 7.27±1.5 3.78±0.04*
20:0 0.11±0.02 0.10±0.01 0.14±0.03 0.07±0.01
22:0 0.06±0.01 0.08±0.01 0.07±0.00 0.07±0.01
∑∑
SFA 40.5±1.1 41.8±2.43 42.05±5.08 40±1.1
16:1 N7 6.99±0.48 8.08±0.7* 5.28±1.4 13.7±0.9*
18:1 N9 23.53±0.9 21.67±1 20.67±0.8 23.55±0.3*
18:1 N7 2.48±0.12 2.4±0.13 2.54±0.15 2.6±0.13
∑∑
MUFA 32.99±0.93 32.14±1.6 28.5±1.5 39.83±0.9*
18:2 N6 23.07±1.4 22.4±0.83 25.6±4 17.33±1.5*
18:3 N6 0.07±0.01 0.06±0.01 0.07±0.01 0.06±0.01
20:3 N6 0.09±0.02 0.10±0.01 0.15±0.02 0.08±0.01*
20:4 N6 0.8±0.15 0.93±0.2 1.0±02 0.7±0.1
22:4 N6 0.61±0.27 0.65±0.25 0.55±0.12 0.44±0.12
∑∑
N6 PUFA 24.54±1.7 24.00±1.05 27.2±4.2 18.52±1.7
18:3 N3 1.5±0.25 1.5±0.21 1.6±0.4 1.2±0.23
20:5 N3 0.04±0.01 0.03±0.01 0.06±0.01 0.03±0.01
22:5 N3 0.1±0.03 0.12±0.03 0.13±0.03 0.09±0.02
22:6 N3 0.23±0.08 0.4±0.08 0.34±0.05 0.25±0.05
∑∑
N3 PUFA 1.86±0.3 1.97±0.31 2.11±0.5 1.54±0.3
GUFA 26.5±1.9 26.1±1.34 29.5±4.7 20.2±1.95
N6/N3=PUFA6/3 12.86±1.95 11.91±1.05 13.06±0.84 13.02±1.6
(18:0/16:0)x100 (elongase) 17±1 16±8 23±3 11±0.3*
(16:1 N7/16:0)x100 (∆9-desaturase) 22±1 25±3 17±4 41±2*
20:4 N6/20:3 N6 (∆5 desaturase) 8±0.7 9±0.5 8±1 9±0.4
20:3 N6/18:2 N6 (∆6 desaturase) 4±0.5 5±0.4 5±0.4 5±0.2
the absolute content (not shown) of saturated fatty
acids. The composition of MUFAs was unchanged af-
ter day 2 of glucose infusion when compared to con-
trol rat, whereas an increase was seen in GR muscle
after 5 days of glucose infusion (~1.4-fold of control).
The increase was particularly obvious for palmitoleic
acid (16:1 N7) (2.6-fold). No changes were seen in the
composition of PUFAs with exception of the ω6 fatty
acids (18:2 N6) and (20:3 N6), which were decreased
in GR after 5 days of glucose infusion (Table 3).
In parallel, the index of ∆9-desaturase was in-
creased 2.3-fold and 3.5-fold after day 2 and day 5 of
glucose infusion, respectively (2 day: GR 14±1 vs
C 6±0.8; 5 day: GR 14±2 vs C 4±1). No changes were
seen in the activity indices of ∆6-desaturase and
elongase whereas the index of ∆5-desaturase was in-
creased by 1.6-fold after day 2 (GR 18±2 vs C 11±1).
In the cholesterol ester fraction, which was very
low in rat skeletal muscle (<10% of triglyceride con-
tent), we found no significant changes in any of the
fatty acids measured (data not shown).
Enzyme activity indices of the fatty acid metabolism.
The determination of the composition of the lipid frac-
tion of skeletal muscles showed that glucose infusion
specifically influenced the content of different fatty
acids, however, palmitoleic acid (16:1 N7) was in-
creased in all lipid fractions. From the summarised ac-
tivity indices of the enzymes necessary for the pro-
cessing of fatty acids (Table 4) it seems that glucose
oversupply did not affect ∆5- and ∆6-desaturases in
most lipid fractions studied (in two fractions <two-
fold) while the activity indices for ∆9-desaturase were
increased more than two-fold after day 2 (in three out
of four lipid fractions) and 2.4-fold to six-fold (in all
four lipid fractions) after day 5 of glucose infusion.
These data indicate that glucose oversupply specifical-
ly enhances ∆9-desaturase activity.
Effect of continuous glucose infusion on
∆
9-desatu-
rase mRNA expression and enzyme activity in rat skel-
etal muscle. A rapid turnover rate has been recently
shown for the microsomal enzyme ∆9-desaturase [32].
208 B. Houdali et al.: Glucose oversupply increases ∆9-desaturase expression and its metabolites in rat skeletal muscle
Table 4. Indices of enzyme activities of elongase and different
desaturases (desat.) Effects of continuous glucose infusion on
FA metabolising enzyme activities in rat skeletal muscle.
Enzyme activity indices were calculated by forming the ratio
of the corresponding product/substrate using results shown in
tables 2 and 3 and in the text. Activities are expressed as x-fold
increase relating to control animals,n=5. Important changes are
indicated in bold
Elongase ∆9-desat. ∆5-desat. ∆6-desat.
day 2 day 5 day 2 day 5 day 2 day 5 day 2 day 5
PL n.s. n.s. 26n.s. n.s. n.s. n.s.
TG n.s. 0.5 n.s. 2.4 n.s. n.s. n.s. n.s.
DG n.s. 0.7 2.1 4.3 n.s. 1.6 n.s. n.s.
NEFA n.s. n.s. 2.3 3.5 1.6 n.s. n.s. n.s.
PL=phospholipid, TG=triglyceride, DG=diglyceride, NEFA
Fig. 2A, B. Time-dependent effect of glucose infusion on ∆9-
desaturase mRNA contents in rat skeletal muscle. (A) RNA
was extracted from skeletal muscle of control (open bars) and
glucose-infused (filled bars) rats and ∆9-desaturase mRNA
were measured by RT-PCR using real time. Controls were set
at 1 and GR were shown as x-fold of increase of controls.
Data represent results of five independent experiments; means
± SEM; ***p<0.001. (B) Representative post run agarose elec-
trophoresis showing the size of ∆9-desaturase PCR-product.
RT-PCR was monitored in real-time using the Light Cycler on-
line monitoring. Post run PCR-products were collected from
PCR capillaries and loaded on 2% agarose gel. Electrophoresis
was run, gel was placed on UV-transilluminator and photo-
graphed using Medidoc gel documentation system (Herolab,
Wiesloch, Germany)
To assess if the effect of glucose oversupply on
MUFA is caused by an increase in ∆9-desaturase turn-
over, the content of ∆9-desaturase mRNA was mea-
sured in rat skeletal muscle by real time RT-PCR. As
shown in Fig. 2A we observed a 10-fold and 75-fold
increase in ∆9-desaturase mRNA content after day 2
and day 5 in skeletal muscle of glucose infused rats,
respectively. The size of the PCR products were eval-
uated by agarose electrophoresis (Fig. 2B). All PCR
products showed the expected size after finishing real
time PCR. These data indicate that glucose oversupply
induces the expression of ∆9-desaturase mRNA.
To further verify the specific induction of ∆9-desat-
urase in skeletal muscle, enzyme activity studies were
carried out as described. In concomitance to the calcu-
lated activity indices ∆9-desaturase enzyme activities
were increased 2.6-fold and 7.7-fold after 2 and
5 days of glucose infusion, respectively (Fig. 3).
Discussion
This study was designed to investigate the relationship
between glucose oversupply, insulin resistance and
lipid composition in rat skeletal muscle. The results
describe the effects of 2 and 5 days of glucose infu-
sion, a condition previously described to cause insulin
resistance in rat skeletal muscle, on the composition
of FA in different lipid fractions. The main findings of
this study are that prolonged glucose infusion induced
(i) specific changes in the composition of the fatty
acids in phospholipids, NEFA, triglycerides and di-
glycerides, ie. the relative content of palmitoleic acid
(16:1 N7) increased several-fold in all lipid fractions
(ii) a striking increase in ∆9-desaturase mRNA ex-
pression and concomitant but lower increase in en-
zyme activity after day 2 and day 5. Furthermore, glu-
cose oversupply led to a decrease in the relative con-
tent of some PUFA; in fact, GUFA were decreased in
nearly all lipid fractions, however, in most cases the
reduction failed to reach statistical significance. The
measured increase in total triglyceride content con-
firms earlier results obtained with this animal model
[8].
Our finding that increasing ∆9-desaturase mRNA
concentrations are higher than the corresponding ∆9-
desaturase enzyme activities are well in line with pre-
vious results [32, 33]. The differences could be ex-
plained by the fact that the half-life of ∆9-desaturase
is very short [32] thus rapid changes of mRNA levels
are not equivalently translated into a corresponding
increase in ∆9-desaturase enzyme activity. It is impor-
tant to note that ∆9-desaturase activity estimated in
microsomes isolated from frozen tissues is lower than
in fresh tissue.
While glucose oversupply obviously increased
palmitoleic acid content in the intermediates of the
lipid metabolism, ie. NEFA and diacylglycerides, the
effect on triglycerides, the storage lipid, was lower
after 2 days but more pronounced after 5 days. The
glucose oversupply also affected the composition of
phospholipids, which play an important functional
role in membranes. Changes in the FA composition of
phospholipids might alter membrane fluidity and per-
meability as was shown by the “leaky membrane” hy-
pothesis [34], and probably diminishes insulin sensi-
tivity due to altered insulin receptor number, reduced
insulin binding capacity and/or altered insulin receptor
tyrosine kinase activity [35, 36, 37, 38] or even more
due to altered post receptor signalling [39, 40, 41, 42].
Using the same samples as in this study we found, that
in rat skeletal muscle early steps of insulin signalling
(phosphorylation of the insulin receptor, IRS-1 and
protein kinase B and the IRS-1-associated phosphati-
dyl-inositol-3′-kinase activity) are inhibited after 2
and particularly after 5 days of glucose infusion[30].
Activation of PKC has been implicated in the devel-
opment of insulin resistance [8, 43, 44, 45, 46]. Of
note, we found an activation of PKC, particularly the
isoform β, in skeletal muscle of glucose infused rats
[30]. Whether an activation of PKC could be related
to the altered FA composition in GR and to the ∆9-de-
saturase induction remains to be shown.
Our data propose some links between glucose over-
supply induced insulin resistance and specific changes
in the FA composition of skeletal muscle lipids. The
findings of a fast and specific increase of palmitoleic
and oleic acid together with an enhanced expression
of ∆9-desaturase indicate that skeletal muscle con-
tains, in addition to the previously shown rapidly reg-
B. Houdali et al.: Glucose oversupply increases ∆9-desaturase expression and its metabolites in rat skeletal muscle 209
Fig. 3. Time-dependent effect of glucose infusion on ∆9-desat-
urase enzyme activity in rat skeletal muscle. Protein was ex-
tracted from skeletal muscle of control (open bars) and glu-
cose-infused (filled bars) rats and specific activity of ∆9-desat-
urase was measured in the microsomal. Data represent results
of three independent experiments; means ± SEM; **p<0.01;
*p<0.05
ulated lipolysis [38], a rapidly and specifically regu-
lated FA metabolism. Our results support the sugges-
tion that altered FA composition in lipid membranes
of skeletal muscle in insulin resistant, obese or diabet-
ic rodents and humans might be due to different desat-
uration availability of the different FA rather than al-
tered fatty acid uptake [13] since different fatty acids
seem not to compete for tissue entry or esterification
[47].
Recent reports show that the gene expression of
∆9-desaturase is highly regulated (reviewed in [48]).
The finding that the half-life time of ∆9-desaturase is
very short indicates that ∆9-desaturase may be a key
regulatory enzyme in lipid metabolism [32]. High car-
bohydrate and insulin induce the hepatic expression of
∆9-desaturase [32, 48]. In rat liver microsomes ∆9-de-
saturase can be induced more than 50-fold by the
administration of a fat-free high-carbohydrate diet.
Abrupt termination of the dietary regimen causes rap-
id decrease of the ∆9-desaturase activity and the pro-
tein content to very low amounts [49]. Little seems to
be known about the regulation of ∆9-desaturase in
skeletal muscle, the main target tissue of insulin-stim-
ulated glucose uptake. In particular, it has been shown
that ∆9-desaturase activity is increased in skeletal
muscle of obese Pima Indians and that this increase is
independently correlated with insulin sensitivity and
obesity in these subjects [50]. We report on the in vivo
regulation of ∆9-desaturase in insulin-resistant skele-
tal muscle of GR, ie. increased MUFAs and increased
∆9-desaturase activity index in nearly all lipid frac-
tions of GR. Our findings that ∆9-desaturase mRNA
expression and enzyme activities were substantially
increased after 2 and 5 days in skeletal muscle argues
for a local desaturation process independent of a pos-
sible enhanced synthesis of MUFA in liver with sub-
sequent transport to muscle. Of note, we observed im-
portant ∆9-desaturase mRNA contents and ∆9-desatu-
rase activities in myotubes obtained from human skel-
etal muscles supporting our findings (E. Schleicher,
unpublished observation). Our results suggest a corre-
lation of high ∆9-desaturase activity with obesity,
muscle insulin resistance and possibly diabetes. Our
data add to the recent discovery of the key role of ∆9-
desaturase in metabolism and energy balance [51].
Previous reports have indicated that elongase activ-
ity (18:0/16:0) is reduced in insulin resistance [16, 50,
52, 53]. In this study, we found that elongase activity
was decreased in day 5 as assessed by the activity in-
dices found in triglycerides and diglycerides fractions.
These data are in accordance with recent reports
showing decreased elongase activity (increased C16:0
on the costs of C18:0) in muscle of healthy subjects
made insulin resistant [7]. In obesity and insulin resis-
tance states, ∆5-desaturase activity was reported to be
reduced [4, 16, 52, 54], whereas that of ∆6-desaturase
increased [16]. We found little change in ∆5-desatu-
rase and no change in ∆6-desaturase activity indices.
In accordance to our findings no changes in the activi-
ties of ∆5- and ∆6-desaturases were found when insu-
lin resistance was induced in normal subjects [7].
In conclusion, continuous glucose infusion into
rats, which causes insulin resistance, leads to an in-
creased accumulation of MUFA in membrane pho-
spholipids and in the storage lipid triglyceride in skel-
etal muscle. The increases in MUFA could be caused
by the strikingly increased ∆9-desaturase activity. Our
results indicate a key regulatory role of this enzyme in
lipid metabolism of skeletal muscle in insulin resistant
states. Although a causal role of ∆9-desaturase in in-
sulin resistance remains to be shown, increased con-
centrations of palmitoleic acid (16:1N7) may serve as
a marker in insulin resistance of skeletal muscle.
Acknowledgements. This study was supported by the German
Diabetes Foundation (75/01/99), the Fortüne Program F 1284126
(University of Tübingen) and by Roche Diagnostics, Mannheim,
Germany. The authors acknowledge the skillful technical assis-
tance of A. Rettig and R. Werner.
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