Received for publication, December 3, 2002, and in revised form, January 9, 2003
Multiple studies suggest that lipid oversupply to
skeletal muscle contributes to the development of insulin resistance,
perhaps by promoting the accumulation of lipid metabolites capable of inhibiting signal transduction. Herein we demonstrate that exposing muscle cells to particular saturated free fatty acids (FFAs), but not
mono-unsaturated FFAs, inhibits insulin stimulation of Akt/protein
kinase B, a serine/threonine kinase that is a central mediator of
insulin-stimulated anabolic metabolism. These saturated FFAs
concomitantly induced the accumulation of ceramide and diacylglycerol, two products of fatty acyl-CoA that have been shown to accumulate in
insulin-resistant tissues and to inhibit early steps in insulin signaling. Preventing de novo ceramide synthesis negated
the antagonistic effect of saturated FFAs toward Akt/protein kinase B. Moreover, inducing ceramide buildup recapitulated and augmented the
inhibitory effect of saturated FFAs. By contrast, diacylglycerol
proved dispensable for these FFA effects. Collectively these results
identify ceramide as a necessary and sufficient intermediate linking
saturated fats to the inhibition of insulin signaling.
 |
INTRODUCTION |
The peptide hormone insulin stimulates the uptake and storage of
glucose in skeletal muscle and adipose tissue while simultaneously inhibiting its efflux from the liver. In certain pathological conditions, including Type 2 diabetes mellitus (1) and metabolic syndrome X (2), these tissues become resistant to insulin such that a
maximal dose of the hormone is unable to elicit these anabolic responses. Numerous studies suggest that the oversupply of lipid to
peripheral tissues might contribute to the development of this insulin
resistance. First, insulin-resistant subjects frequently display signs
of abnormal lipid metabolism including obesity (3), increased
circulating free fatty acid
(FFA)1 concentrations (4, 5),
and elevated intramyocellular lipid levels (6). In fact, the size of
the intramyocellular lipid depot correlates more tightly with the
severity of insulin resistance than most known risk factors (6).
Second, experimentally exposing peripheral tissues to lipids decreases
their sensitivity to insulin. For example, (a) incubating
isolated muscle strips or cultured muscle cells with FFAs (7-11),
(b) infusing lipid emulsions into rodents or humans
(12-15), or (c) expressing lipoprotein lipase in skeletal
muscle of transgenic mice (16, 17) promotes intramyocellular lipid
accumulation and compromises insulin-stimulated glucose uptake. These
observations have prompted investigators to hypothesize that increased
availability of lipids to peripheral tissues causes insulin resistance,
perhaps by promoting the accumulation of one or more fat-derived
metabolites capable of inhibiting insulin action (6, 18).
The insulin receptor is a heterotetrameric tyrosine kinase receptor
that mediates all of the anabolic effects of insulin (19). The
activated receptor phosphorylates intracellular docking molecules (termed insulin receptor substrates, or IRS proteins) that recruit and
stimulate multiple different effector enzymes (20).
Phosphatidylinositol (PI) 3-kinase is a target of IRS proteins that is
an obligate intermediate in the metabolic, anti-apoptotic, and
mitogenic effects of insulin (21). PI 3-kinase phosphorylates specific
phosphoinositides to generate phosphatidylinositol-3,4-bisphosphate,
and phosphatidylinositol-3,4,5-trisphosphate, which subsequently
recruit cytosolic serine/threonine kinases phosphoinositide-dependent kinase-1 and Akt/protein kinase
B (PKB) to the plasma membrane. The association between these
phosphoinositides and the Akt/PKB pleckstrin homology domain promotes
Akt/PKB activation by facilitating its phosphorylation on two
regulatory residues (i.e. Ser-473 and Thr-308) (22). Studies
in cultured cells involving the overexpression of constitutively active
(23) or dominant negative (24, 25) forms of Akt/PKB coupled with
experiments involving the microinjection of inhibitory anti-Akt/PKB
antibodies (26) indicate the involvement of the enzyme in the
regulation of anabolic metabolism. Moreover, knockout mice lacking the
Akt2/PKB
isoform develop a diabetes-like syndrome characterized by
insulin resistance in both skeletal muscle and liver (27).
The molecular mechanisms linking FFAs to the inhibition of insulin
action remain unclear. In 1963, Randle et al. (28) proposed the existence of a glucose-fatty acid cycle by which glucose and lipids
could serve as competitive substrates for oxidation in muscle. More
recent studies in either cultured cells (9-11) or rodent models of
obesity and/or insulin resistance (29, 30), however, indicate that
fatty acids also disrupt one or more early steps in insulin signal
transduction. As shown herein, the saturated fats palmitate and
stearate, but not their mono-unsaturated counterparts oleate and
palmitoleate, blocked insulin activation of Akt/PKB while concomitantly
promoting the accumulation of ceramide and diacylglycerol in C2C12
myotubes. These lipid metabolites have both been shown to accumulate in
tissues from insulin-resistant rodents (31) and to inhibit insulin
signal transduction in cultured cells (32-37). Specifically, studies
with short chain ceramide analogs reveal that ceramide prevents insulin
activation of Akt/PKB (32-34), whereas investigations with phorbol
esters, which mimic the effects of diacylglycerol (DAG), indicate that
DAG blocks upstream signaling events by promoting the serine
phosphorylation of IRS-1 (38-42). Using various methods for
manipulating either the synthesis or breakdown of ceramide, we found
that endogenously produced ceramide was both sufficient and necessary
for the inhibition of insulin signaling by palmitate. Moreover, we
determined that DAG was dispensable for the inhibitory palmitate
effects. These findings implicate ceramide as a potentially important
intermediate linking saturated fats to the development of insulin resistance.
 |
EXPERIMENTAL PROCEDURES |
Reagents--
C2-ceramide and
DL-threo-1-Phenyl-2-decanoylamino-3-morpholino-1-propanol
(PDMP) were obtained from Calbiochem, okadaic acid was from Invitrogen,
fetal bovine serum was from Atlas Biologicals (Fort Collins, CO), and
silica gel 60 thin layer chromatography (TLC) plates were from Merck.
The following additional reagents were obtained from Sigma: palmitate,
stearate, oleate, palmitoleate, Dulbecco's modified Eagle's medium
(DMEM), fatty acid free bovine serum albumin, C-6 ceramide, C-16
ceramide, C-18 ceramide, fumonisin B1, myriocin, cycloserine, and
N-oleoylethanolamine). Antibodies utilized included rabbit
polyclonal anti-phospho-Akt (Ser-473) and anti-phospho-glycogen
synthase kinase 3
(GSK3
) (serine 9) antibodies from Cell
Signaling, mouse anti-glycogen synthase kinase 3
(GSK3
) antibody
from Transduction Labs (Lexington, KY), rabbit anti-phospho-mitogen-activated protein kinase antibody from Promega (Madison, WI), rabbit polyclonal anti-Akt, and horseradish
peroxidase-conjugated anti-rabbit and anti-mouse antibodies from Santa
Cruz Biotechnology (Santa Cruz, CA).
Cell Culture--
C2C12 myoblasts were maintained at 37 °C in
DMEM containing 10% fetal bovine serum. For differentiation into
myotubes, the myoblasts were grown to confluency, and the medium was
replaced with DMEM containing 10% horse serum. Myotubes were used for
experiments 4 days after differentiation.
FFA Treatment--
Free fatty acids were administered to cells
by conjugating them with FFA-free bovine serum albumin. Briefly, FFAs
were dissolved in ethanol and diluted 1:100 in 1% FBS-DMEM containing
2% (w/v) bovine serum albumin. Two hours before performing the
experiments, myotubes were placed in serum free-DMEM containing 2%
bovine serum albumin in either the presence or absence of FFAs.
Immunoblot Analysis--
Cell lysates were resolved by SDS-PAGE,
transferred to nitrocellulose, and immunoblotted using methods
described previously (43). Detection was done using the Enhanced
Chemiluminescence or the Enhanced Chemiluminescence Plus kit from
Amersham Biosciences according to the manufacturer's instructions.
Phosphatidylinositol 3-Kinase Activity--
PI 3-kinase activity
was assessed using the methodology of Summers et al.
(44).
Ceramide Assay--
Myotubes were lysed in ice-cold 1 M NaCl (0.25 ml) and transferred to glass tubes. Ice cold
CHCl3/CH3OH (1:2, v/v, 0.75 ml) was added, and
the suspension was vortexed vigorously. After the subsequent addition
of 0.25 ml of 1 M NaCl and 0.25 ml of CHCl3, phases were separated by centrifugation at 3000 rpm (2000 × g) for 5 min. Ceramide content in the extract was determined
using a radiometric diacylglycerol assay kit (Amersham Biosciences) according to the manufacturer's instructions.
Adenovirus Amplification, Titration, and
Infection--
Small T cDNA served as a template for PCR
amplification. The upstream primer (5'-AGATCTATGGATAAAGTTTTAAACAG-3')
incorporated a BglII site and an ATG start codon, and the
downstream primer (5'-CTCGAGTTAGAGCTTTAAATCTCT-3') was engineered with
a XhoI site. The amplified products were cloned directly
into TOPO2.1 (Invitrogen) and sequenced in both directions to confirm
that no errors were introduced by PCR. The small T TOPO2.1 cDNA was
digested with BglII and XhoI, and the approximate
0.5-kilobase small T fragment was gel-purified and subcloned into the
pAdTrack-CMV shuttle vector (45). Recombinant adenoviruses were
generated by homologous recombination after electroporation of small T
with pAdEASY-1 into RecA+ bacteria (BJ5183). Bacterial
clones containing recombinant adenoviral DNA were verified by
restriction digestion with PacI, and recombinant viruses
were generated by transfection of HEK 293 cells. Virus was amplified by
four rounds of infection and purified from 20 × 15-cm plates of
HEK 293 cells using two rounds of centrifugation through CsCl
gradients. The residual CsCl was removed by dialysis against 10%
glycerin in phosphate-buffered saline in a Slide-a-Lyzer cassette.
Virus was titered using the methods of Minamide et al. (45),
and myotubes were infected at a multiplicity of infection of 100. Under
these conditions, greater than 80% of the myotubes expressed the green
fluorescent protein protein, which is also encoded by the recombinant
adenovirus. Experiments were performed 48 h after infection.
 |
RESULTS |
To identify specific FFAs capable of antagonizing insulin signal
transduction, we evaluated the effect of specific saturated and
monounsaturated FFAs on insulin-stimulated Akt/PKB phosphorylation and
activation in C2C12 myotubes. Palmitate (C16:0) and stearate (C18:0),
which comprise greater than 90% of the saturated FFAs in human serum
(46), each markedly inhibited insulin stimulation of Akt/PKB
phosphorylation (Fig. 1A). By
contrast, neither oleate (C18:1), which makes up 80% of the
circulating monounsaturated pool (46), nor palmitoleate (C16:1), had
any effect (Fig. 1A). Palmitate and stearate also inhibited
insulin-stimulated phosphorylation of GSK3
(Fig. 1A), a
substrate of Akt/PKB with numerous functions including the regulation
of glycogen synthase activity (44). Palmitate inhibited Akt/PKB
phosphorylation within 2-4 h (Fig. 1B) at a FFA
concentration as low as 0.25 mM (Fig. 1C). This
concentration is comparable with that found physiologically (9) and is
similar to that used in prior studies evaluating FFA effects in both
immortalized muscle cells (9, 10) and isolated skeletal muscle strips (11). Neither palmitate nor stearate inhibited IRS1-associated PI
3-kinase (Fig. 2).

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Fig. 1.
Palmitate and stearate inhibit Akt/PKB
phosphorylation (P) and activation. A,
C2C12 myotubes were incubated in the presence or absence of the
indicated FFAs (16 h, 0.75 mM) before stimulation with
insulin (Ins, 100 nM, 10 min). Cell lysates were
resolved by SDS-PAGE, transferred to nitrocellulose, and immunoblotted
with the indicated antibodies. Detection was by enhanced
chemiluminescence. Data are representative of three independent
experiments. B, C2C12 myotubes were incubated with palmitate
(0.75 mM) for the time indicated before analysis as in
A above. Data are representative of three independent
experiments. C, C2C12 myotubes were incubated with palmitate
(16 h) at the concentration indicated before analysis as in
A above. Data are representative of three independent
experiments.
|
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Fig. 2.
Palmitate and stearate do not inhibit
IRS1-associated PI 3-kinase activity. C2C12 myotubes were
incubated in the presence or absence of the indicated fatty acids (16 h, 0.75 mM) before stimulation with insulin
(Ins, 100 nM, 10 min). IRS-1 was
immunoprecipitated from cell lysates and incubated with
[32P]ATP and phosphoinositide. Lipids were extracted and
resolved by thin layer chromatography. Phosphoinositide 3-phosphate
(PI(3)P) produced by the phosphorylation of phosphoinositide
by PI 3-kinase was visualized using a Storm PhosphorImager. Data
are representative of three independent experiments.
|
|
We next determined whether the inhibitory FFAs also induced the
accumulation of DAG and/or ceramide. Briefly, DAG kinase can phosphorylate both DAG and ceramide to produce phosphatidic acid and
ceramide 1-phosphate, respectively, which can be resolved by TLC. When
the reaction is allowed to proceed in the presence of
[32P]ATP, the phosphorylated products can be detected
using a Storm PhosphorImager (47). As shown in Fig.
3, palmitate and stearate induced
ceramide accumulation ~3-fold over basal levels (Fig. 3A),
whereas they induced DAG accumulation ~6- and 3.5-fold, respectively (Fig. 3B). By contrast, neither oleate nor palmitoleate had
any effect on either ceramide or DAG accumulation. Interestingly, another saturated FFA, myristate (14:0), induced DAG synthesis while
having no effect on ceramide accumulation or Akt/PKB or GSK3
phosphorylation (data not shown).
As described earlier, ceramide blocks insulin signaling by preventing
the activation of Akt/PKB (9, 32), whereas DAG inhibits "upstream"
signaling events (i.e. insulin stimulation of
IRS-1-associated PI 3-kinase (38-42)). Because palmitate also inhibited Akt/PKB but not PI 3-kinase (Fig. 2), we hypothesized that
ceramide and not DAG was the principal effector linking saturated FFAs
to the inhibition of Akt/PKB. To test this hypothesis, we determined
whether inhibitors of de novo ceramide synthesis could prevent palmitate inhibition of insulin signaling. Briefly, ceramide biosynthesis requires the coordinate action of two enzymes,
i.e. serine palmitoyltransferase and ceramide synthase (for
review, see Ref. 48). Serine palmitoyltransferase catalyzes the initial step, which involves the condensation of serine and palmitoyl-CoA to
form 3-ketosphinganine, a sphingolipid that is subsequently reduced to
form the sphingoid base sphinganine. Ceramide synthase catalyzes
sphinganine acylation, producing dihydroceramide, which is then
converted to ceramide by the introduction of a trans-4,5 double bond in the sphinganine moiety. Pretreating C2C12 myotubes with
myriocin, a fungal toxin that inhibits serine palmitoyltransferase, completely prevented the palmitate-induced increase in ceramide levels
(Fig. 4A) but had no effect on
DAG accumulation (Fig. 4B). As predicted, myriocin
completely negated the inhibitory effect of palmitate on both Akt/PKB
and GSK3
phosphorylation (Fig.
5A). Similarly, cycloserine,
another serine palmitoyltransferase inhibitor, also reversed the
palmitate effect on Akt/PKB phosphorylation (Fig. 5B).
Fumonisin B1, a fungal toxin that inhibits ceramide synthase, also
prevented the palmitate effect on ceramide (Fig. 4A), but
not DAG (Fig. 4B), accumulation. Like myriocin and
cycloserine, fumonisin B1 protected both Akt/PKB and GSK3
from the
inhibitory effects of palmitate (Fig. 5C). Myriocin,
cycloserine, or fumonisin B1 did not affect the expression or the basal
or insulin-stimulated phosphorylation of either Akt/PKB or GSK3
(data not shown). Moreover, myriocin and fumonisin B1 did not prevent
the inhibition of Akt/PKB phosphorylation by exogenous C2-ceramide
(data not shown).
Most of the work revealing that ceramide inhibits Akt/PKB relies on the
use of short chain ceramide analogs, which are questionable in terms of
their ability to mimic endogenous ceramide (49). Moreover, both
C2-ceramide and endogenously produced ceramide can be rapidly
deacylated and glucosylated to give rise to a broad array of
sphingolipid-derived molecules (for review, see Ref. 48). Thus, two
questions remained. First, could endogenous ceramide mimic the effects
of both C2-ceramide and palmitate; second, is ceramide itself or,
alternatively, another ceramide metabolite, the principal mediator of
the inhibitory effects of palmitate. To address this issue, we treated
cells with inhibitors of ceramide glucosylation (i.e. the
glucosylceramide synthase inhibitor PDMP) or deacylation
(i.e. the ceramidase inhibitor
N-oleoylethanolamine). These compounds were shown previously
to increase endogenous ceramide levels by blocking its normal route(s)
of metabolism (50). Treating C2C12 myotubes with either compound
increased cellular ceramide to levels comparable with those achieved
with palmitate alone (Fig.
6A), and both drugs markedly
inhibited insulin-stimulated Akt/PKB phosphorylation (Fig.
6B). Neither drug affected the expression of Akt or GSK3
(data not shown). Thus, increasing endogenous ceramide levels by an
alternative mechanism recapitulated the inhibitory effects of palmitate
on Akt/PKB phosphorylation. We next attempted to determine whether
these drugs, by blocking ceramide glucosylation or deacylation, could
actually augment the palmitate effect on either the accumulation of
long chain ceramides or the antagonism of insulin signaling. As
predicted, the inclusion of either PDMP or
N-oleoylethanolamine along with palmitate potentiated the
effects of either reagent individually on both ceramide accumulation (Fig. 6A) and the inhibition of Akt/PKB phosphorylation
(Fig. 6, C and D).
Ceramide has been shown to prevent Akt/PKB activation by at least two
independent mechanisms. In certain cell types, okadaic acid, an
inhibitor of protein phosphatase 2A (PP2A), negates the antagonistic
effects of ceramide on Akt/PKB. This finding suggests that ceramide
blocks insulin action by accelerating the rate of Akt/PKB
dephosphorylation (34, 51, 52). In other cell types, however, okadaic
acid has no effect, and ceramide instead blocks the insulin-stimulated
translocation of Akt/PKB to the plasma membrane (32, 33). To test
whether the effects of palmitate required PP2A in C2C12 myotubes, we
pretreated cells with okadaic acid before treating with insulin. In the
presence of okadaic acid, palmitate did not prevent insulin-stimulated
Akt/PKB phosphorylation (Fig.
7A). However, because okadaic
acid stimulated Akt/PKB phosphorylation in the absence of insulin we
could not distinguish between (a) okadaic acid reversing the
effect of palmitate or (b) okadaic acid stimulation of
Akt/PKB being insensitive to ceramide. To more definitively determine
whether palmitate was working through PP2A, we used recombinant
adenovirus to overexpress the SV40 small T antigen. This protein was
shown previously to inhibit PP2A activity by displacing one of the
regulatory subunits of the enzyme (53). SV40 small T expression
completely prevented the effects of both palmitate and C2-ceramide on
Akt/PKB phosphorylation (Fig. 7B), confirming the likely
involvement of PP2A in the inhibitory effects of saturated FFAs in
C2C12 myotubes.
The strong correlation between intramyocellular triglyceride
levels and the severity of insulin resistance suggests that lipid oversupply to peripheral tissues could cause or exacerbate the condition. Many researchers have hypothesized that one or more derivatives of fatty acyl-CoA are likely to link inappropriate fat
deposition in skeletal muscle to the inhibition of insulin signaling
(6). Possible products of long chain acyl-CoAs capable of inhibiting
insulin signaling include ceramide and DAG (for review, see Ref. 18),
but a definitive role for neither has been established. The data
presented herein indicate that endogenously produced ceramide is both
capable of inhibiting Akt/PKB and, more importantly, is necessary for
the inhibitory effects of saturated FFAs. First, palmitate, stearate,
and C2-ceramide blocked insulin signaling at the same step
(i.e. by blocking activation of Akt/PKB but not stimulation
of PI 3-kinase (Figs. 1 and 2)) (9). Second, inhibiting de
novo ceramide synthesis completely prevented palmitate induction
of ceramide synthesis and its antagonism of Akt/PKB and GSK3
phosphorylation (Figs. 4 and 5). Third, preventing ceramide metabolism
and/or degradation recapitulated the effects of palmitate on both
intracellular ceramide accumulation and the inhibition of insulin
signaling (Fig. 6). Fourth, blocking ceramide metabolism while
concomitantly adding palmitate augmented its effect on both ceramide
accumulation and the inhibition of Akt/PKB phosphorylation (Fig. 6).
And fifth, expressing the SV40 small T antigen, an inhibitor of PP2A,
prevented the effects of both C2-ceramide and palmitate on Akt/PKB
phosphorylation (Fig. 7). Collectively these data implicate ceramide in
insulin resistance resulting from the oversupply of saturated FFAs to
skeletal muscle.
To manipulate intracellular ceramide levels in these experiments, we
relied on the use of a large number of enzyme inhibitors to block
ceramide synthesis or degradation. To minimize the possibility that our
observations were the result of nonspecific pharmacological effects, we
employed inhibitors capable of blocking separate enzymes in the various
pathways. For example, the fungal toxins myriocin and fumonisin inhibit
separate enzymes that are required for de novo ceramide
synthesis (i.e. serine palmitoyltransferase and ceramide
synthase, respectively) (48), and both protected C2C12 myotubes from
the inhibitory effects of palmitate. Cycloserine, a serine
palmitoyltransferase inhibitor that is structurally dissimilar to
myriocin, also prevented the antagonistic effects of palmitate. These
compounds did not affect Akt/PKB expression or activation in the
absence of FFAs (data not shown), and none were capable of blocking the
antagonistic effects of exogenously added C2-ceramide, which presumably
bypasses the site of action of these inhibitors (data not shown). To
induce ceramide accumulation we also used unique compounds with
separate intracellular targets. Both PDMP and
N-oleoylethanolamine, by inhibiting glucosylceramide
synthase and ceramidase, respectively, were able to induce ceramide
accumulation and to block Akt/PKB activation (Fig. 6). Thus, by using a
broad array of inhibitors, our data conclusively indicate that ceramide is both a necessary and sufficient intermediate linking palmitate to
the antagonism of insulin signaling.
Although ceramide proved to be required for the effects of palmitate,
the DAG derived from saturated FFAs was incapable of inhibiting insulin
signaling to Akt/PKB. Specifically, in Figs. 4 and 5 we demonstrate
conditions where insulin signaling was inhibited despite the fact that
DAG accumulation was not. The hypothesized involvement of DAG in
insulin resistance derives from multiple prior studies both in cultured
cells and rodents. For example, treating various cell lines with
phorbol esters, which mimic the effects of DAG, inhibits insulin
signaling to IRS-1 (38-41) and Akt (42). Moreover, several PKC
isoforms including PKC
(54, 55), -
1 and -
2 (56), -
(38),
-
(55), and -
(38) are downstream effectors of DAG that
purportedly antagonize insulin signaling by phosphorylating IRS-1 on
inhibitory serine residues. Observations that DAG levels are often
elevated in rodent models of insulin resistance (for review, see Ref.
57) have fueled speculation that the lipid may serve as a critical
antagonist of insulin signaling in diseased tissues. The discrepancy
between these studies and those mentioned above may be reconciled by
recent observations that the acyl chain composition in DAG may
participate in its ability to activate PKC isoforms. Specifically, DAG
derived from saturated fatty acids is generally a poor activator of
PKC, whereas that produced from polyunsaturated fatty acyl CoAs is a
much stronger stimulus (58). Thus, DAG may participate in the
regulation of insulin signaling by polyunsaturated FFAs, whereas ceramide is the principle metabolite linking saturated FFAs to insulin signaling.
The mechanism by which ceramide inhibits insulin action is unclear.
Ceramide has been proposed to activate PP2A, the phosphatase responsible for dephosphorylating Akt/PKB in adipocytes (59). Moreover,
in PC12 cells (52), brown adipocytes (34), and human glioblastoma cell
line U87MG (60), the PP2A inhibitor okadaic acid prevents the effects
of short chain ceramide analogs on Akt/PKB phosphorylation. Although
these studies suggest the existence of a mechanism by which
ceramide increases intracellular PP2A activity, several other
reports show that the effects of ceramide are okadaic acid-insensitive.
Specifically, studies in hybrid motor neurons (61), 3T3-L1
pre-adipocytes and adipocytes (32), A7r5 vascular smooth muscle cells
(62), and L6 muscle cells (33) all demonstrate a profound inhibition of
Akt/PKB by C2-ceramide that is insensitive to okadaic acid. In most of
these cell types, ceramide was shown to block the insulin-stimulated
recruitment of Akt/PKB to the plasma membrane. Prior studies by
Cazzolli et al. (63) indicated that the inhibitory
effects of palmitate in C2C12 myotubes were also dependent upon okadaic
acid. However, we found that okadaic acid in fact stimulated Akt/PKB
phosphorylation in the absence of insulin, something shown for other
cell types (59) (Fig. 7A). This observation limited the
usefulness of the drug as a means for dissecting the pathways linking
palmitate to the inhibition of Akt/PKB since one could not distinguish
whether okadaic acid prevented or simply bypassed the site of ceramide action. Expressing the SV40 small T antigen, an inhibitor of PP2A, confirmed the likely involvement of PP2A in the inhibitory pathway linking both palmitate and ceramide to the regulation of Akt/PKB phosphorylation in C2C12 myotubes (Fig. 7B).
Numerous studies implicate saturated fats, particularly palmitate,
in the development of insulin resistance. First, palmitate is one of
the most abundant FFAs found in skeletal muscle as well as being one of
the most prevalent acyl chains found in the diglyceride fraction of
lipid extracts (64). Second, an inverse relationship exists between the
consumption of palmitate and insulin sensitivity (65, 66). And third,
insulin-resistant muscles demonstrate accelerated rates of palmitate
uptake (67). A similar cadre of results suggests that the
over-accumulation of ceramide could participate in the development of
insulin resistance. First, various rodent models of insulin resistance,
including Zucker fa/fa rats (31) and mice overexpressing
lipoprotein lipase (16), demonstrate elevated intramuscular ceramide
levels. Second, exercise training Wistar rats improves insulin
sensitivity while markedly lowering intramuscular ceramide levels (68).
And third, short chain ceramide analogs inhibit insulin-stimulated
Akt/PKB activation and/or glucose uptake in 3T3-L1 adipocytes (32, 35),
brown adipocytes (34), C2C12 and L6 myotubes (9, 33), and isolated rat
skeletal muscle (69). The experiments described herein clearly indicate that endogenously produced ceramide is not only capable of antagonizing insulin action but, more importantly, is required for the inhibitory effects of long chain saturated fatty acids on insulin signaling. Although aberrant ceramide accumulation is unlikely to account entirely
for the diverse array of defects found in insulin resistant tissues,
the findings presented herein implicate ceramide as a potentially
important mediator of the deleterious effects of long chain saturated
fats. Studies attempting to block ceramide synthesis in various animal
models of insulin resistance will be necessary to determine the extent
to which this lipid metabolite contributes to the pathologies
associated with the condition.
We thank Norman Curthoys, Jennifer Nyborg,
and Suzanne Stratford (Colorado State University) for providing
intellectual input into the study design and for critically reviewing
the manuscript before submission.
Published, JBC Papers in Press, January 13, 2003, DOI 10.1074/jbc.M212307200
The abbreviations used are:
FFA, free fatty
acid;
IRS, insulin receptor substrate;
PI, phosphatidylinositol;
PKB, protein kinase B;
DAG, diacylglycerol;
GSK3
, glycogen synthase
kinase 3
;
PDMP, DL-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol;
HEK cells, human embryonic kidney cells;
DMEM, Dulbecco's modified
Eagle's medium;
CMV, cytomegalovirus;
PP2A, protein phosphatase
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