Leptin opposes insulin's effects on fatty acid partitioning
in muscles isolated from obese ob/ob
mice
Deborah M.
Muoio1,
G. Lynis
Dohm2,
Edward B.
Tapscott2, and
Rosalind A.
Coleman1
1 Departments of Nutrition and
Pediatrics, University of North Carolina at Chapel Hill, Chapel
Hill 27599; and 2 Department of
Biochemistry, East Carolina University, Brody Medical Sciences
Building, Greenville, North Carolina 27858
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ABSTRACT |
Because muscle
triacylglycerol (TAG) accumulation might contribute to insulin
resistance in leptin-deficient ob/ob
mice, we studied the acute (60- to 90-min) effects of leptin and
insulin on
[14C]glucose and
[14C]oleate metabolism
in muscles isolated from lean and obese
ob/ob mice. In
ob/ob soleus, leptin decreased
glycogen synthesis 36-46% (P < 0.05), increased oleate oxidation 26%
(P < 0.05), decreased oleate
incorporation into TAG 32% (P < 0.05), and decreased the oleate partitioning ratio (oleate partitioned
into TAG/CO2) 44% (P < 0.05). Insulin decreased oleate
oxidation 31% (P < 0.05), increased
oleate incorporation into TAG 46% (P < 0.05), and increased the partitioning ratio 125%
(P < 0.01). Adding leptin diminished insulin's antioxidative, lipogenic effects. In soleus from lean mice,
insulin increased the partitioning ratio 142%, whereas leptin decreased it 51%, as previously reported (Muoio, D. M., G. L. Dohm, F. T. Fiedorek, E. B. Tapscott, and R. A. Coleman.
Diabetes 46: 1360-1363, 1997).
The phosphatidylinositol 3-kinase inhibitor wortmannin blocked
insulin's effects on lipid metabolism but only attenuated leptin's
effects. Increasing glucose concentration from 5 to 10 mM did not
affect TAG synthesis, suggesting that insulin-induced lipogenesis is
independent of increased glucose uptake. These data indicate that
leptin opposes insulin's promotion of TAG accumulation in lean and
ob/ob muscles. Because acute leptin exposure does not correct insulin resistance in
ob/ob muscles, in vivo improvements in
glucose homeostasis appear to require other long-term factors, possibly
TAG depletion.
fatty acid oxidation; obesity; triacylglycerol
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INTRODUCTION |
LEPTIN, a 16-kDa peptide encoded by the
ob gene, is the first identified
adipocyte-derived hormone that directly regulates adiposity and energy
homeostasis by decreasing food intake and increasing energy expenditure
(30, 47). Homozygous ob/ob mice, which
lack functional leptin, are characterized by severe adiposity, hyperlipidemia, hyperglycemia, and hyperinsulinemia (47), thereby exhibiting an obesity syndrome that is phenotypically similar to type
II diabetes. When administered to
ob/ob mice, leptin reverses obesity
and normalizes serum concentrations of glucose, insulin, and lipids
(30, 39). Leptin affects these serum variables at low doses that do not
affect body weight (30), and improvements in serum variables are
greater in leptin-treated ob/ob mice
than in pair-fed ob/ob controls (22).
These observations indicate that leptin exerts metabolic effects that
are independent of its effects on food intake and body weight.
Leptin receptors (L-Rs) are expressed primarily in the hypothalamus but
are also present in several peripheral tissues, including liver,
pancreas, ovary, kidney, heart, and skeletal muscle (5, 13), suggesting
that leptin may exert direct effects on tissues other than the brain.
Emerging evidence indicates that peripheral L-Rs play an important
physiological role in mediating leptin's regulation of fuel
homeostasis. We reported that leptin controls skeletal muscle lipid
metabolism by partitioning fatty acids (FA) toward oxidation and away
from storage as triacylglycerol (TAG; see Ref. 26). In isolated soleus
muscle from lean C57BL/6J mice, leptin acutely increases oleate
oxidation and decreases oleate incorporation into TAG. In addition, we
reported that leptin attenuates insulin's antioxidative and lipogenic
effects on skeletal muscle lipid utilization. Our data from acute
incubations of skeletal muscle are similar to those obtained from
chronically incubated isolated pancreatic islets in which 3 days of
leptin exposure increased FA oxidation, decreased FA esterification,
and depleted islet cell TAG content (40). These data demonstrate that
leptin directly regulates peripheral lipid metabolism.
Skeletal muscle is a major determinant of whole body glucose and lipid
metabolism and is the primary tissue accountable for reduced whole body
insulin responsiveness in type II diabetes. The pathological mechanisms
underlying type II diabetes are still largely undefined, but
considerable evidence links the development of muscle insulin
resistance with increased muscle lipid content (28, 31). Because in
vivo leptin treatment dramatically improves insulin sensitivity and
depletes muscle TAG in ob/ob mice (22, 30, 40), we studied the acute effects of leptin and its interaction with insulin on glucose metabolism and FA partitioning in muscles isolated from ob/ob mice. Contrary to
our hypothesis that acute leptin exposure would improve
insulin-mediated glucose regulation, we found that leptin decreased
glycogen synthesis in muscles from ob/ob mice. We also report that, in
muscles isolated from ob/ob mice,
insulin promotes TAG accumulation, whereas leptin favors lipid
oxidation and TAG depletion. These data indicate that leptin and
insulin have acute opposing effects on fuel metabolism in muscles from
ob/ob mice and suggest that
leptin-induced improvements in glucose homeostasis observed in vivo may
require centrally mediated effects and/or long-term regulatory factors
such as protein synthesis and muscle TAG depletion.
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RESEARCH DESIGN AND METHODS |
Animals. Female C57BL/6J
ob/ob mice and lean littermates
(Jackson Laboratory, Bar Harbor, ME) were maintained on a 12:12-h light-dark cycle with food (Purina mouse chow) and
H2O available ad libitum. During
the studies of glucose metabolism, Purina chow was removed from the
animal's cages at 1700 on the evening before the experiments. Animals
used for glucose experiments were 8-12 wk old because leptin
improves glucose homeostasis in ob/ob
mice of this age (30, 39). Because older animals are preferred for
studying lipid metabolism, animals used for lipid experiments were
12-18 wk old. The animal protocol was approved by the University of North Carolina Institutional Animal Care and Use Committee.
Muscle incubations. Recombinant murine
ob peptide (leptin) was supplied by
Amgen (Thousands Oaks, CA). Soleus and extensor digitorum longus (EDL)
muscles were removed between 0900 and 1100 under anesthesia (100 mg/kg
ketamine and 10 mg/kg xylazine), cleaned free of adipose and connective
tissue, and immediately transferred to a 24-well tissue culture dish in
a shaking H2O bath at 29°C. Each well contained 1.0 ml of a modified Krebs-Ringer bicarbonate buffer [KRB; low-calcium Krebs Henseleit bicarbonate buffer (8), 1.0% (wt/vol) dialyzed, FA-free BSA, and 5.0 mM glucose] and was gassed continuously with 95%
O2-5%
CO2. After all muscles were isolated, they were preincubated for an additional 15 min at 37°C before being transferred to fresh media (as will be described).
Lipid metabolism. In experiments that
examined hormone effects on lipid partitioning, muscles were
preincubated for an additional 20 min at 37°C in KRB media with 1.5 mM oleate, 1.0 mM carnitine, and 1.5% BSA (KRB + FA).
Muscles were then transferred to fresh KRB + FA containing
[1-14C]oleate (1.0 µCi/ml; American Radio Chemical, St. Louis, MO) and incubated for 90 min. Previous dose-response experiments indicated that, in isolated
muscles, maximal hormone responses are obtained at 10.0 mU/ml insulin
and 10.0 µg/ml leptin (12, 18, 26). Maximal levels of hormone, either
insulin (Eli Lilly, Indianapolis, IN) alone (10.0 mU/ml), leptin alone
(10.0 µg/ml), or insulin and leptin combined, were added to one-half
of the muscles, and the contralateral soleus and EDL muscles from each
mouse were used as basal (non-hormone-treated) controls. Preliminary
experiments indicated that rates of oleate oxidation increased linearly
with 0 to 1.0 mM oleate and were maximal at 1.5 mM oleate (data not shown). To inhibit lipolysis of intramuscular TAG and thereby minimize
label dilution effects that may result from intramuscular lipid
hydrolysis (29, 43), 1.0-1.5 mM oleate was used in all experiments
studying lipid metabolism.
In experiments to test the effects of wortmannin on leptin and
insulin-mediated regulation of FA partitioning, incubations were
performed as described above, but one muscle from each animal was used
as a basal (non-hormone-treated) control, and contralateral solei were
incubated for 90 min at 37°C in the presence of either insulin
(10.0 mU/ml), wortmannin (1.0 µM), or insulin plus wortmannin; or
leptin (10.0 µg/ml) or leptin plus wortmannin. To determine whether
increasing the media glucose concentration mimics the effects of
insulin on muscle FA partitioning, soleus muscles from lean C57BL/6J
mice were incubated, as described in the previous paragraph, in media
containing 5.0 or 10 mM glucose, in the presence or absence of insulin
(10 mU/ml).
After the incubation, muscles were washed two times in ice-cold KRB for
10 min, weighed, and immediately frozen at
80°C. Substrate
oxidation was quantified using a modified
CO2 trapping technique (14) in
which semidry filter paper (Whatman no. 3) saturated with 2 N NaOH was
placed over the 24-well incubation dish and tightly covered with a foam
pad and the plate cover. [14C]CO2
produced by muscle was driven from the media to the filter paper trap
by adding 100 µl of 70% perchloric acid to each well. After 60 min
of trapping in a shaking bath at 37°C, the filter paper disks
corresponding to each well were dried, cut, and washed in 2.0 ml
distilled H2O.
[14C]CO2
was quantified in 1.0-ml aliquots of the
H2O wash by liquid scintillation
counting. Increases in 14C over
background were minimal in media in which muscles had been incubated,
indicating that insignificant amounts of acid-soluble metabolites had
been released in the medium (14). Lipids were extracted from muscle,
and TAG, diacylglycerol (DAG), phospholipid, and FA were separated by
thin-layer chromatography on scored silica gel G plates (Fisher
Scientific, Norcross, GA) in hexane-diethyl ether-acetic acid (80:20:1,
vol/vol/vol). Radioactivity incorporation into different lipid species
was determined by comparison with standards using a Bioscan 200 system.
Carbohydrate metabolism. In
experiments that examined hormone effects on glucose metabolism,
muscles were preincubated for an additional 20 min at 37°C in KRB,
transferred to fresh KRB containing
[U-14C]glucose
(1.0 µCi/ml; Sigma Chemical, St. Louis, MO), and incubated for 60 min. Maximal levels of hormone, either insulin alone (10.0 mU/ml),
leptin alone (10.0 µg/ml), or insulin and leptin combined, were added
to one-half of the muscles, and the contralateral soleus and EDL
muscles from each mouse were used as basal (non-hormone-treated) controls. Immediately after the incubation, semidry filter paper (Whatman no. 3) saturated with 2 N NaOH was placed over the 24-well incubation dish, and
[14C]CO2
produced by muscle was trapped and quantified as described in Lipid
metabolism as an indicator of glucose oxidation. After the
CO2 trapping procedure, muscles
were removed from the wells, weighed, and extracted in KOH to
precipitate glycogen (12). Radioactivity incorporated into glycogen was
measured by liquid scintillation counting. Lactate concentrations in
neutralized medium were assayed enzymatically (9).
Statistics. Data are presented as
means ± SE and were analyzed by a one-way ANOVA followed by
comparison using Newman-Keuls post hoc test. Because muscles from
ob/ob mice exhibited significant interanimal variation in basal fuel metabolism as well as in hormone responsiveness, differences between basal and hormone-treated contralateral muscles were also analyzed by paired Student's
t-test.
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RESULTS |
Leptin inhibits glycogen synthesis.
Body weights of obese ob/ob mice (53 ± 7.4 g) were two times those of lean littermates (25 ± 3.8 g).
In soleus and EDL muscles from lean animals, insulin increased glycogen
synthesis by eight- and sixfold, respectively (Fig.
1A).
Compared with muscles from lean mice, insulin-stimulated increases in
glycogen synthesis were ~50% lower in muscles from ob/ob mice
(P < 0.01), consistent with
obesity-associated insulin resistance. In soleus muscles from
ob/ob mice, leptin inhibited glycogen
synthesis by 35% (P < 0.05; Fig.
1B); adding insulin abolished
leptin's inhibition. In EDL, leptin diminished glycogen synthesis in
both the absence and presence of insulin by 38 and 42%
(P < 0.05), respectively. These data
differ from our previous observations using muscles from lean mice in
which leptin did not alter basal or insulin-stimulated glycogen
synthesis (26). Similar to muscles from lean mice (26), leptin did not
affect glucose oxidation or lactate production in
ob/ob muscles incubated with or
without insulin (data not shown).

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Fig. 1.
Insulin-stimulated glycogen synthesis in muscles isolated from
ob/ob mice and lean littermates.
Soleus and extensor digitorum longus (EDL) muscles from lean and
ob/ob mice were isolated and incubated
in 1.0 ml of a modified Krebs-Ringer media containing 5.0 mM glucose.
Then, one muscle from each animal was used as a basal
(non-hormone-treated) control, and the contralateral soleus and EDL
muscles were incubated with 1.0 µCi/ml
[U-14C]glucose for 60 min at 37°C with insulin (10.0 mU/ml;
A) and with or without leptin (10.0 µg/ml; B).
[14C]glucose
incorporation into glycogen was measured as described in RESEARCH
DESIGN AND METHODS. Data are means ± SE of 5-8
muscles/group and were analyzed by unpaired Student's
t-test.
P < 0.01, statistically
significant differences between lean and
ob/ob muscles;
* P < 0.05 and
** P < 0.001, differences
between hormone-treated groups.
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Leptin and insulin regulate FA oxidation in
ob/ob muscles. Basal
rates of oleate oxidation in ob/ob
muscles did not differ from those in muscles from lean mice (Table
1). Compared with contralateral basal
controls, insulin inhibited the rate of oleate oxidation in soleus by
31% and in EDL by 23% (Fig.
2A;
P < 0.05). In contrast, leptin
increased the rates of oleate oxidation in both soleus and EDL muscles
by 26 and 20%, respectively (P < 0.05). When leptin and insulin were present together, leptin completely blocked insulin's antioxidative effect.
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Table 1.
Basal rates of oleate oxidation and esterification into TAG in
muscles from ob/ob mice and lean littermates
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Fig. 2.
Effects of leptin and insulin on muscle fatty acid (FA) oxidation and
FA incorporation in triacylglycerol (TAG). Intact soleus and EDL
muscles from ob/ob mice were incubated
in 1.0 ml of modified Krebs-Ringer media containing 1.5 mM oleate and
5.0 mM glucose. Then, one muscle from each animal was used as a basal
(non-hormone-treated) control (n = 15 muscles), and the contralateral soleus and EDL muscles
(n = 5/group) were incubated for 90 min at 37°C in the presence of either insulin (10.0 mU/ml), leptin
(10.0 µg/ml), or insulin plus leptin. Incubation media contained 1.0 µCi/ml of
[1-14C]oleate.
[14C]oleate
incorporation into CO2
(A) and muscle TAG
(B) was determined as described in
RESEARCH DESIGN AND METHODS. Data (means ± SE) are
expressed as percent change in hormone-treated muscles compared with
nontreated contralateral controls (basal values are provided in text).
Data were analyzed by paired Student's
t-test.
* P < 0.05 and
** P < 0.01, statistically
significant differences compared with basal values.
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Leptin and insulin regulate esterification of FA into
TAG in ob/ob muscles.
Under basal conditions the rates of FA incorporation into TAG were
similar in muscles from ob/ob and lean
mice (Table 1). Hormone effects on FA esterification were opposite of
their effects on FA oxidation (Fig.
2B). In soleus muscle, insulin
stimulated the esterification of FA into TAG by 47%
(P < 0.01), whereas leptin decreased FA incorporation into TAG by 32%
(P < 0.05). In EDL, neither leptin
nor insulin affected incorporation into TAG.
Leptin and insulin have opposing effects on muscle
lipid partitioning in ob/ob
muscles. FA entering muscle can be esterified and
incorporated into complex lipids (primarily TAG) to provide the muscle
with an endogenous energy reservoir, or FA can be oxidized to provide
an immediate source of energy. The partitioning of FA between these two
fates largely determines the TAG content of muscle. To quantify the
partitioning of FA between biosynthetic and oxidative pathways, we
divided the nanomoles per gram muscle per hour of FA esterified into
TAG by the nanomoles per gram muscle per hour of FA oxidized to
CO2, providing a ratio of lipid
stored to lipid oxidized (Fig. 3). In
soleus and EDL muscles the ratio of FA stored to FA oxidized was ~4:1
and 2:1, respectively, under basal conditions. The divergent effects of
each hormone on FA oxidation compared with FA storage resulted in
marked changes in muscle lipid partitioning. In soleus muscle, insulin
increased this ratio by 125%, whereas leptin decreased it by 44%
(P < 0.01 and
P < 0.05). When muscles were
coincubated with both hormones, leptin blocked the insulin-induced
increase in the ratio of FA incorporated into
TAG/CO2. EDL exhibited similar,
but less pronounced, hormone-mediated changes in FA partitioning. These
data indicate that, in muscles from
ob/ob mice, insulin promotes TAG
accumulation, whereas leptin favors TAG depletion.

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Fig. 3.
Effects of leptin and insulin on muscle FA partitioning. Intact soleus
(A) and EDL
(B) muscles from
ob/ob mice were incubated in 1.0 ml of
modified Krebs-Ringer media containing 1.5 mM oleate and 5.0 mM
glucose. Then, one muscle from each animal was used as a basal
(non-hormone-treated) control (n = 15 muscles), and the contralateral soleus and EDL muscles
(n = 5/group) were incubated for 90 min at 37°C in the presence of either insulin (10.0 mU/ml), leptin
(10.0 µg/ml), or insulin plus leptin. Incubation media contained 1.0 µCi/ml of
[1-14C]oleate. Label
incorporation into CO2 muscle TAG
was determined as described in RESEARCH DESIGN AND METHODS.
Data (means ± SE) are expressed as a ratio of
[14C]oleate
incorporated into TAG
(nmol · g 1 · h 1)
divided by label incorporated into
CO2
(nmol · g 1 · h 1).
Data were analyzed by one-way ANOVA.
* P < 0.05 and
** P < 0.01, significant
differences between basal and hormone-treated muscles;
P < 0.05, differences
between insulin alone and other hormone treatments.
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Insulin affects FA incorporation into other complex
lipids. In muscles isolated from lean mice, insulin
increased 14C label incorporation
into DAG by 42 and 72% in soleus and EDL, respectively (Fig.
4A).
Similarly, in soleus muscles isolated from
ob/ob mice, insulin increased FA
incorporation in DAG by 100% (Fig.
4B). When insulin was present, the
rate of oleate incorporation into DAG was greater in
ob/ob than in lean soleus. Table
2 shows the fractions of
[14C]oleate that were
incorporated into major acylglycerol species recovered in muscles
isolated from lean and ob/ob mice. In
both soleus and EDL, most of the labeled oleate was present as TAG, but
the percentage of label incorporated into TAG, DAG, and phospholipid was different (P < 0.01) between the
two muscle types. Soleus stored a higher percentage of FA as TAG than
did EDL. These data, together with those in Fig. 2B showing
that the rate of FA incorporation into TAG was higher in soleus than
EDL, are consistent with previous reports in lean animals showing that
highly oxidative muscles have a greater TAG content than glycolytic
muscles (24). Insulin did not affect the percentage of FA
incorporated into TAG in soleus and EDL from either lean or
ob/ob mice. However, in soleus and EDL
from ob/ob mice, insulin increased the
percentage of FA incorporated into DAG
(P < 0.05). In soleus from both lean
and ob/ob mice, insulin decreased the
proportion of label incorporated into phospholipid (P < 0.05). Adding leptin to the
media did not alter the fractions of FA label incorporated into
different lipid species, nor did it affect the absolute amount of
labeled DAG (data not shown).

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Fig. 4.
Effects of insulin on muscle FA incorporation into diacylglycerol
(DAG). Intact soleus and EDL muscles from lean
(A) and
ob/ob
(B) mice were incubated in 1.0 ml of
modified Krebs-Ringer media containing 1.5 mM oleate and 5.0 mM
glucose. Then, one muscle from each animal was used as a basal
(non-hormone-treated) control (n = 5),
and the contralateral soleus and EDL muscles
(n = 5) were incubated for 90 min at
37°C in the presence of insulin (10.0 mU/ml). Incubation media
contained 1.0 µCi/ml of
[1-14C]oleate.
14C label incorporation into
muscle DAG was determined as described in RESEARCH DESIGN AND
METHODS, and data are presented as means ± SE.
* P < 0.05 and
** P < 0.01, significant
differences between basal and contralateral insulin-treated muscles by
paired Student's t-test.
P < 0.05, comparisons
between lean and ob/ob muscles by
unpaired Student's t-test.
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Insulin's effects on FA partitioning are mediated by
phosphatidylinositol 3-kinase. Insulin-mediated
regulation of muscle glucose metabolism requires activation of
phosphatidylinositol 3-kinase (PI 3-kinase; see Refs. 21 and 46). PI
3-kinase may also be involved in leptin signaling (6). To determine
whether PI 3-kinase mediates insulin and/or leptin-induced alterations in muscle lipid partitioning, we incubated solei from wild-type animals
with insulin or leptin, alone or in the presence of the PI 3-kinase
inhibitor wortmannin. Similar to insulin's effect on lipid metabolism
in muscles from ob/ob mice (Fig. 2),
in muscles from lean mice, insulin decreased FA oxidation by 50% and
increased FA esterification into TAG by 65%
(P < 0.01; Fig.
5, A-C).
Although wortmannin alone did not affect muscle FA metabolism,
wortmannin completely blocked insulin's inhibition of FA oxidation and
insulin's stimulation of FA esterification. Leptin increased FA
oxidation by 43% and decreased FA incorporation into TAG by 31%
(P < 0.01), as we reported
previously (26). Wortmannin prevented the leptin-induced increase in FA
oxidation but did not block the leptin-mediated decrease in FA
esterification into TAG (Fig. 5,
D-E). Thus wortmannin only partially
blocked leptin's effects on muscle FA partitioning (Fig.
5F).

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Fig. 5.
Effects of wortmannin on insulin and leptin-mediated regulation of
muscle FA partitioning. Intact soleus and EDL muscles from lean
C57BL/6J mice were isolated and incubated in 1.0 ml of modified
Krebs-Ringer media containing 1.5 mM oleate and 5.0 mM glucose. After
preincubation, one muscle from each animal was used as a basal (B;
non-hormone-treated) control (n = 9),
and the contralateral soleus muscles
(n = 6) were incubated for 90 min at
37°C in the presence of either insulin (I; 10.0 mU/ml), wortmannin (W; 1.0 µM), or insulin plus
wortmannin (I + W; A-C);
or leptin (L; 10.0 µg/ml), wortmannin (W; 1.0 µM), or leptin plus
wortmannin (L + W; D-F).
Incubation media contained 1.0 µCi/ml of
[1-14C]oleate. Label
incorporation into CO2 and muscle
TAG and the ratio TAG/CO2 were
determined as described in RESEARCH DESIGN AND METHODS.
Data (means ± SE) were analyzed by one-way ANOVA.
* P < 0.05, significant
differences compared with basal values;
P < 0.05, significant
differences between leptin and leptin plus wortmannin.
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Glucose competes with FA as a substrate for oxidation and, upon
entering the glycolytic pathway, provides glycerol 3-phosphate for de
novo glycerolipid biosynthesis. Thus, because wortmannin prevents
insulin-stimulated glucose uptake (21, 46), wortmannin's inhibition of
insulin-mediated FA partitioning (Fig. 5,
A-C) might suggest that insulin's
effects are secondary to increased glucose uptake. To test the effect
of glucose uptake directly, we incubated muscles from lean mice with
and without insulin in the presence of increasing glucose
concentrations to increase insulin-independent glucose uptake. In the
absence of insulin, increasing the glucose concentration from 5 to 10 mM decreased FA oxidation by 24 and 28%
(P < 0.05) in soleus and EDL
muscles, respectively, but had no effect on FA incorporation into TAG
(Fig. 6). Adding insulin decreased FA
oxidation by 37-40% and increased FA incorporation in TAG by
57-66% in soleus and EDL muscles. Doubling the media glucose
concentration from 5 to 10 mM did not, however, augment insulin's
antioxidative or lipogenic effects. These data suggest that an
insulin-mediated increase in glucose uptake could inhibit muscle FA
oxidation but is not responsible for the increase in FA esterification.

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Fig. 6.
Effects of media glucose concentration on muscle FA oxidation and FA
incorporation in TAG. Intact soleus and EDL muscles from lean C57BL/6J
mice were isolated and incubated in 1.0 ml of modified Krebs-Ringer
media containing 1.5 mM oleate and either 5.0 or 10.0 mM glucose. After
preincubation, one muscle from each animal was used as a basal
(non-hormone-treated) control (n = 4-6), and the contralateral soleus muscles
(n = 4-6) were incubated for 90 min at 37°C in the presence of insulin (10.0 mU/ml). Incubation
media contained 1.0 µCi/ml of
[1-14C]oleate. Label
incorporation into CO2
(A) and muscle TAG
(B) was determined as described in
RESEARCH DESIGN AND METHODS. Data (means ± SE) were
analyzed by unpaired Student's
t-test.
* P < 0.05, statistically
significant differences between muscles incubated in 5.0 vs. 10.0 mM
glucose.
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DISCUSSION |
In isolated soleus and EDL muscles from
ob/ob mice, leptin and insulin had
acute and opposing effects on muscle lipid metabolism; leptin favored
lipid oxidation, whereas insulin favored muscle lipid storage as TAG.
Consistent with our previous report on leptin's effects on muscles
from lean mice (26), hormone-induced alterations in muscle lipid
metabolism were more pronounced in soleus, a highly oxidative muscle,
than EDL, a more glycolytic muscle.
Our data provide additional evidence that leptin's effects on energy
metabolism are partly mediated by the hormone's direct actions on
peripheral tissues (6, 10, 22, 39). Alternative splicing of the L-R
gene provides at least five unique L-R mRNA isoforms that encode
proteins that differ in the length of their intracellular domains and
that are expressed in multiple tissues at varying amounts (4, 44). The
L-R isoform with a long intracellular domain is highly expressed in the
hypothalamus and is believed to be primarily responsible for mediating
leptin-induced signal transduction (13); however, short L-R isoforms
might also transduce a leptin signal (17, 27). In skeletal muscle and
other peripheral tissues, the majority of L-R mRNAs expressed encode
for the short forms of the receptor (5, 10, 13, 44). The functions of
the different L-R isoforms have not been established, but our data
provide evidence that peripheral L-Rs mediate acute alterations in
muscle lipid metabolism and suggest that the peripheral actions of
leptin complement its central actions.
Similar effects of leptin on FA partitioning in isolated pancreatic
islets were observed after chronic incubation with leptin (25). In
isolated islets incubated for 48 h, leptin increases mRNA
abundance for enzymes that promote FA oxidation: acyl-CoA oxidase,
carnitine palmitoyltransferase, and uncoupling protein-2 (49).
Furthermore, in these islets, leptin decreases mRNA abundance for
enzymes involved in lipogenesis, such as acetyl-CoA carboxylase and
mitochondrial glycerol-3-phosphate acyltransferase. In vivo leptin
treatments increase mRNA expression of skeletal muscle uncoupling
protein-3, a mitochondrial protein that is upregulated in response to
starvation and exercise, physiological states in which muscle FA
oxidation is enhanced (38, 45). These changes in mRNA abundance might
explain why chronic, in vivo leptin treatments deplete the TAG content
of adipose tissue, liver, pancreas, and skeletal muscle (25), but it is
unclear whether the effects observed during our 90-min incubations
could have resulted from alterations in gene expression.
Because leptin injections given to
ob/ob mice dramatically improve
glucose metabolism and insulin sensitivity, we hypothesized that leptin
might directly increase insulin-stimulated glycogen synthesis in
muscle. Instead, we found that leptin has no effect on glucose
metabolism in muscles from lean animals (26), whereas in
ob/ob muscles leptin decreased
glycogen synthesis, similar to a previous report (23). Other in vitro
studies have demonstrated that leptin has no direct effects on basal or
insulin-stimulated glucose uptake in muscles isolated from either lean
or ob/ob mice (50). Similarly, in
rats, short-term in vivo leptin administration had no effect on muscle
glycogen synthesis or 2-deoxyglucose uptake (34). Another report
suggests that muscle responses to leptin may depend on previous leptin
exposure. In C57BL/6J mice, leptin injections acutely (2 h) decreased
whole body glucose clearance, but, after chronic (7 days) treatment,
leptin increased insulin-stimulated muscle glycogen synthesis in vivo
and increased soleus muscle glucose uptake in vitro (16). Taken
together, these observations indicate that improvements in glucose
homeostasis reported in leptin-treated
ob/ob mice are mediated by long-term
regulatory factors requiring chronic leptin exposure. Furthermore,
leptin's effects on energy regulation occur largely through centrally
mediated neuroendocrine responses that increase sympathetic output,
thermogenesis, and energy expenditure and decrease food intake and body
weight (1). Thus leptin-regulated adjustments in glucose homeostasis and insulin sensitivity might also require central factors that are
absent from in vitro preparations.
The etiology of muscle insulin resistance is complex and still largely
undefined. Muscle lipid accumulation appears to be linked to insulin
resistance. For example, obesity and type II diabetes are strongly
associated with increased muscle TAG and DAG content, and decreased FA
oxidation (3, 7, 15, 28, 31, 42) and reversal of insulin resistance are
associated with depletion of muscle lipid (28, 35). It has been
hypothesized that perturbed muscle lipid metabolism, leading to TAG
accumulation, contributes to the pathogenesis of insulin resistance
(15, 31). The mechanism(s) by which lipid accumulation could interfere
with glucose metabolism might involve alterations in lipid modulators of insulin-signaling pathways, such as DAG and acyl-CoA (32, 36). We
found that, even though muscles from
ob/ob mice were insulin resistant,
when insulin was added to the media,
ob/ob muscles preferentially
partitioned FA toward TAG and away from
-oxidation, thereby
increasing the ratio of FA partitioned into TAG/CO2 by more than twofold
compared with basal muscles. Data from this study and our earlier
report (26) are the first to show that insulin suppression of FA
oxidation is directly associated with a concomitant increase in the
amount of FA incorporated into neutral lipid and that insulin regulates
muscle FA partitioning, even in obese, insulin-resistant states. In
human studies, hyperglycemic, hyperinsulinemic clamping markedly
reduces whole body FA oxidation, but effects on muscle TAG have not
been reported. Although the concentrations of hormones used in this
study were 10- to 100-fold higher than in serum, others have
demonstrated that these relative doses are required to elicit maximal
hormone responses in incubated skeletal muscle (12, 18, 26).
Supraphysiological hormone concentrations may be required because the
muscle fiber architecture does not permit optimal exposure of receptors
to hormones.
Insulin's effect on muscle FA partitioning appears to be mediated by
activation of PI 3-kinase, since it was completely blocked by
wortmannin. Wortmannin also blocks insulin-stimulated glucose uptake
(21, 46), suggesting that insulin's effects on FA partitioning might
be secondary to insulin-stimulated glucose uptake. Alternatively, insulin's regulation of muscle lipid metabolism could be due to PI
3-kinase-mediated inactivation of oxidative enzymes and/or activation
of lipogenic enzymes. Our observation that increasing the media glucose
concentration decreased muscle FA oxidation suggests that acute insulin
suppression of muscle FA oxidation is at least partly due to increased
glucose uptake. Increased glucose uptake results in increased muscle
concentrations of citrate, which allosterically activates acetyl-CoA
carboxylase and stimulates the production of malonyl-CoA (37).
Malonyl-CoA, a key inhibitor of FA oxidation, is thus the likely link
between insulin-stimulated glucose uptake and insulin inhibition of FA
oxidation. In contrast, insulin's stimulatory effect on FA
esterification in muscle lipids is likely mediated by acute activation
of one or more of the enzymes involved in glycerolipid biosynthesis,
since increasing the media glucose concentration in the absence of
insulin did not augment TAG synthesis. Furthermore, these data suggest
that the low rates of FA oxidation and the increased concentrations of
TAG previously reported in muscle from obese animals (3, 35) and humans (7, 15) were probably due in part to chronic hyperinsulinemia. Because
we found that leptin attenuated insulin's antioxidative and lipogenic
actions, leptin's ability to improve insulin sensitivity in vivo might
be partly mediated by its inhibition or reversal of muscle TAG
accumulation (40).
It has been suggested that leptin and insulin are counterregulatory
hormones that regulate energy homeostasis (33). In isolated rat
adipocytes, leptin diminishes insulin-stimulated glucose uptake and
attenuates insulin's antilipolytic and lipogenic effects (25). In
isolated pancreatic islets and in perfused pancreas, leptin inhibits
insulin secretion, and, in Hep G2 hepatocytes, leptin inhibits
insulin-induced decreases in
phosphoenolpyruvate carboxykinase expression (6). We found that, in isolated muscle, leptin opposes the
antioxidative and lipogenic effects of insulin. The ability of leptin
to attenuate insulin's actions on peripheral glucose and lipid
metabolism is consistent with observations that leptin modulates the
phosphorylation of insulin-signaling proteins. In Hep G2 hepatocytes,
leptin downregulates insulin-dependent phosphorylation of insulin
receptor substrate (IRS)-1 (6). Similarly, in Rat1 fibroblasts, leptin
impairs phosphorylation of the insulin receptor and of IRS-1 (19).
Leptin may not, however, oppose insulin action in every tissue. For
example, in cultured
C2C12
myotubes, which express only the short L-R, leptin mimicked insulin's
actions by stimulating glucose uptake and glycogen synthesis through a PI 3-kinase-dependent pathway (2, 17). Likewise, in Hep G2 cells,
leptin increases PI 3-kinase activity and its association with IRS-1,
and in pancreatic islets leptin-induced activation of phosphodiesterase
3B is also mediated by PI 3-kinase (48). These observations, together
with our data showing that wortmannin attenuated leptin's effects on
muscle lipid partitioning, suggest that activation of PI 3-kinase might
be common to both the leptin and insulin-signaling cascades. These
observations also imply that, in some peripheral tissues, leptin and
insulin-signaling pathways may converge, and, furthermore, that the
interactions between leptin and insulin are tissue specific and may be
related to the expression pattern of distinct L-R isoforms.
Muscles from ob/ob mice are likely to
contain more intermuscular fat than muscle from lean animals. Because
any adipose tissue present would contribute minimally to total
oxidation, the presence of intermuscular fat should not affect FA
oxidation. If hormonal effects on TAG synthesis were due largely to
contaminating adipose tissue, one would expect that the effects would
be greater for ob/ob muscle (which
contains more intermuscular fat). Instead, our data showed effects on
TAG synthesis that were similar or greater in lean compared with
ob/ob muscle, suggesting that the results were due to the regulation of lipogenesis in muscle, rather than adipose tissue.
 |
ACKNOWLEDGEMENTS |
We thank Amgen for donating the leptin for this study.
 |
FOOTNOTES |
This research was supported, in part, by the National Institutes of
Health (NIH) Grants HD-19068 (to R. A. Coleman) and DK-38416 (to G. L. Dohm) and the North Carolina Institute of Nutrition. D. M. Muoio was
supported by a Traineeship from the NIH (DK-07686), the North Carolina
American Heart Association, and a University of North Carolina
Dissertation Fellowship.
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for correspondence and reprint requests: R. A. Coleman, Dept.
of Nutrition, Univ. of North Carolina at Chapel Hill, 2209 McGavran-Greenberg, CB #7400, Chapel Hill, NC 27599 (E-mail:
rcoleman{at}sph.unc.edu).
Received 16 October 1998; accepted in final form 20 January 1999.
 |
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