Garvan Institute of Medical Research, Sydney, New South Wales 2010, Australia
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ABSTRACT |
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We have recently shown that the reduction in
insulin sensitivity of rats fed a high-fat diet is associated with the
translocation of the novel protein kinase
C
(nPKC
) from cytosolic to
particulate fractions in red skeletal muscle and also the
downregulation of cytosolic
nPKC
. Here we have further
investigated the link between insulin resistance and PKC by assessing
the effects of the thiazolidinedione insulin-sensitizer BRL-49653 on
PKC isoenzymes in muscle. BRL-49653 increased the recovery of nPKC
isoenzymes in cytosolic fractions of red muscle from fat-fed rats,
reducing their apparent activation and/or downregulation,
whereas PKC in control rats was unaffected. Because BRL-49653 also
improves insulin-stimulated glucose uptake in fat-fed rats and reduces
muscle lipid storage, especially diglyceride content, these results
strengthen the association between lipid availability, nPKC activation,
and skeletal muscle insulin resistance and support the hypothesis that
chronic activation of nPKC isoenzymes is involved in the generation of
muscle insulin resistance in fat-fed rats.
insulin resistance; thiazolidinedione
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INTRODUCTION |
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REDUCED WHOLE BODY RESPONSE to insulin is a major feature of type II, or non-insulin-dependent diabetes mellitus, and is mainly attributable to a decrease in insulin-stimulated glucose uptake by skeletal muscle (17). Although the mechanism by which this muscle insulin resistance arises is unclear, several animal and human studies have suggested a strong link with increased tissue lipid availability (7, 23, 25, 38). For example, in the well-characterized high-fat-fed rat model of insulin resistance, which does not feature substantial hyperglycemia and hyperinsulinemia, increases in skeletal muscle triglyceride and diglyceride (DG) levels accompany the diminished capacity for insulin-stimulated glucose disposal (27).
Derivatives of triglyceride, especially DG, are activators of the protein kinase C (PKC) family of signal transduction enzymes (26). In keeping with this, we have recently shown that chronic activation and/or downregulation of specific PKC isoenzymes accompanies decreased insulin sensitivity of muscle from fat-fed rats and correlates well with muscle triglyceride and DG levels (34). Alterations in DG and PKC have also been observed in other models of insulin resistance (2, 16, 19, 21), and, because these enzymes have been shown to inhibit insulin signaling in vitro, we and others have hypothesized that chronic activation of one or more PKC isoenzymes is involved in the generation of this disorder (11, 15, 34, 36). This hypothesis should be distinguished from the controversial role of PKC in normal insulin action (5, 20). Possible targets for phosphorylation, and hence inhibition, by PKCs include early components of the insulin signal transduction pathway, especially the adaptor molecule insulin receptor substrate-1 (IRS-1) (14), as well as metabolic enzymes such as glycogen synthase (1).
The PKC family can be divided into three groups on structural and
functional bases (see Ref. 26 for review): the conventional PKC
isoenzymes ,
, and
, which are dependent on calcium, DG, and
phospholipid for activity; the novel (n)PKC isoenzymes
,
,
,
and
, which are calcium independent; and the atypical PKC isoenzymes
and
, which are independent of both calcium and DG. Activation
of PKC in the presence of these lipids occurs at cell membranes, and
hence a reduction in cytosolic PKC and its recovery in membrane
fractions are frequently taken as a measure of activation of the kinase
(26). Consequently, the decreased proportion of
nPKC
observed in the cytosolic
fraction of muscles from fat-fed rats, relative to starch-fed controls
(34), suggests that activation of this isoenzyme accompanies the
reduction in insulin sensitivity. Furthermore, chronic activation of
PKC isoenzymes can lead to their proteolysis (12, 41). Thus in the red
muscle of fat-fed rats there is a reduction in the total levels of
nPKC
, apparent only in the
cytosolic component (34), which is suggestive of the downregulation of
this kinase, possibly after prolonged stimulation.
The current studies were undertaken to test whether the changes in cytosolic nPKC levels in muscles from fat-fed rats would be reversed by the insulin-sensitizing compound BRL-49653, which reduces systemic lipid supply and utilization (28). This prediction follows from the hypothesis that chronic alterations in lipid activators and hence PKC activity in muscle lead to a reduction in insulin sensitivity. Our results confirm that manipulation of insulin sensitivity, by fat feeding and drug treatment, is associated with alterations in the levels and distribution of specific nPKC isoforms.
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MATERIALS AND METHODS |
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Materials.
Rabbit antipeptide antibodies against PKCs ,
,
,
, and
were from GIBCO BRL, Life Technologies, Mulgrave, Australia. Rabbit
antipeptide antibody against
nPKC
was from Santa Cruz
Biotechnology, Santa Cruz, CA. Horseradish peroxidase-linked donkey
anti-rabbit antibody was from Jackson Immuno Research Laboratories, West Grove, PA. Renaissance-enhanced chemiluminescence reagents were
from NEN, Boston, MA. Other biochemicals were mostly from Sigma
Chemical or BDH Laboratory Supplies.
Experimental animals and dietary treatment.
All experimental procedures performed for this study were approved by
the Animal Experimentation Ethics Committee (Garvan Institute) and were
in accordance with the National Health and Medical Research Council of
Australia guidelines on animal experimentation. Procedures were carried
out as described previously (28). Briefly, male Wistar rats weighing
~250 g were fed isocaloric rations (350 kJ/day) of either a
high-starch or a high-fat diet for 3 wk up to the study day. The
composition of the fat diet was 59% fat, 21% protein, and 20%
carbohydrate (38); the starch diet consisted of 10% fat, 21% protein,
and 69% carbohydrate (23). Starch- and fat-fed rats were divided into
BRL-49653-treated and control subgroups. Treated rats were given four
doses of BRL-49653 (10 mmol · kg1 · day
1)
at 0730 by gastric gavage, commencing 3 days before and finishing on
the day of killing. Control rats were gavaged with an equal volume of
vehicle (saline). Body weights of rats after 3 wk of diet feeding were
347.5 ± 5.0 (starch fed, untreated), 348.2 ± 1.5 (starch fed,
treated), 378.5 ± 4.7 (fat fed, untreated) and 376.2 ± 4.2 (fat
fed, treated), n = 6 in each group.
Tissue extraction and immunoblotting.
Red gastrocnemius and red quadriceps muscles were collected rapidly
after animals were killed by pentobarbitone overdose, frozen with
liquid N2-cooled tongs, and stored
at 80°C. Muscle cytosolic and solubilized-particulate
fractions were prepared as described previously (34) and subjected to
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (~50 and 25 mg protein, respectively). Proteins were electroblotted onto
nitrocellulose membranes, which were probed with rabbit antipeptide
antibodies specific for PKC isoenzymes
,
,
,
, or
,
followed by horseradish peroxidase-linked donkey anti-rabbit antibody
(34). PKC isoenzymes were then visualized by incubation of the membrane
with enhanced chemiluminescence reagents and exposure to X-ray film for
between 30 s and 10 min. Densitometry of PKC bands was carried out
using a Medical Dynamics Personal Densitometer SI and analyzed using IP
Lab Gel H software (Signal Analytics, Vienna, VA). The relationship between the amount of sample subjected to immunoblotting and the signal
intensity observed was linear under the conditions described above. As
previously, the ratio of the dry weight of the detergent-insoluble fraction to the wet weight of the starting material was used to correct
for variations in recovery between samples (34). This measurement
exhibited low variability and was not significantly different between
starch- and fat-fed rats [e.g., 5.48 ± 0.08 (n = 6) vs. 5.41 ± 0.04 (n = 6), respectively, in one
experiment].
Presentation of results. To study the effects of BRL-49653 on PKC, four groups of rats were examined: starch-fed untreated, starch-fed drug treated, high-fat-fed untreated, and high-fat-fed drug treated. The mean cytosolic PKC content of starch-fed untreated rat muscle was set to 100% in each case, and the mean cytosolic PKC of muscle in the other groups was expressed relative to this. This is the optimal measurement, since any change in either the location or total content of a specific nPKC in the muscles was clearly reflected in the cytosolic component. Indeed, isoenzymes that exhibited downregulation did so only in cytosolic fractions, whereas membrane-associated levels remained unchanged (34). Furthermore, cytosolic PKC measurements showed less variability between samples from the same group than those of membrane-associated PKC, most likely because they were obtained after fewer experimental procedures that could affect recovery. For the estimation of total PKC content, cytosolic and membrane amounts were added. We have previously determined that only traces of PKC isoenzymes are detected in the detergent-insoluble fraction (34), and these were not quantitated.
Statistics. All results are expressed as means ± SE. Analysis of diet and treatment effects was by unpaired Student's t-test. Statistical calculations were performed using Statview SE + GraphicsTM for Macintosh (Abacus Concepts, Berkeley, CA).
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RESULTS |
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PKC and atypical
PKC
are not affected by fat
feeding in skeletal muscle (34), but we included measurements of these
isoenzymes in the study to assess more fully the effects of BRL-49653
on the PKC family. No significant effects of diet or BRL-49653
treatment were observed on either
PKC
(Fig. 1A, diet
effect P > 0.25, drug effect
P > 0.75, both muscle types combined) or PKC
(Fig.
1B, diet effect
P > 0.70, drug effect P > 0.65, both muscle types
combined), which therefore serve as useful negative controls when
considering changes in other PKC isoenzymes.
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There was a fall in cytosolic levels of
nPKC in both red quadriceps and
red gastrocnemius muscles of untreated rats in response to fat feeding
(Fig.
2A),
confirming previous results (34). Although total amounts did not change
(Fig. 2B), there was also a
corresponding rise in membrane-associated levels, consistent with a
translocation of this isoenzyme (Fig.
2C). Treatment with BRL-49653 did
not significantly affect cytosolic
nPKC
in either muscle from
starch-fed rats but was able to partly reverse the fall in cytosolic
levels seen in muscle from fat-fed rats (Fig.
2A, P < 0.01 for fat-fed treated vs. fat-fed untreated, both muscle types
combined). As a consequence, the ratio of membrane-associated to
cytosolic nPKC
in muscle from
these rats fell by over 30% (Fig.
2C), so that the alteration due to
fat feeding was overcome by ~50%.
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Fat feeding also caused a decrease in the amount of cytosolic
nPKC in both muscles (Fig.
3A), but
this was not accompanied by changes in membrane-associated kinase,
which meant that total levels of this isoenzyme were diminished in
muscles from fat-fed rats (Fig. 3B),
whereas the ratio of membrane-associated to cytosolic protein increased
(Fig. 3C). When rats were treated
with BRL-49653, no change was again observed in the cytosolic levels of
nPKC
in muscles from starch-fed
rats (Fig. 3A). However, the
decrease seen in muscles from fat-fed rats was partially reversed (Fig. 3A; P < 0.002 for fat-fed treated vs. fat-fed untreated, both muscle types
combined). This was also apparent in the total levels of
nPKC
in muscle from fat-fed
rats, which were increased by 55% on treatment with the drug (Fig.
3B). The ratio of
membrane-associated to cytosolic
nPKC
therefore also fell (Fig.
3C).
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Similar results were also seen in the case of
nPKC (Fig.
4), in that cytosolic and hence total
levels of this kinase fell in muscles from fat-fed rats, although the
changes were less pronounced than those exhibited by
nPKC
. Treatment with BRL-49653
tended to elevate cytosolic
nPKC
in fat-fed rats, which was
best demonstrated as a decrease in the ratio of membrane to cytosolic
levels of the isoenzyme (Fig. 4C).
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DISCUSSION |
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This study was designed to test further the hypothesis that chronic activation of PKC isoenzymes in red skeletal muscle is associated with diminished insulin sensitivity, since this would help to provide a mechanistic explanation of the link between increased availability and metabolism of lipid and the generation of insulin resistance. By employing a high-fat diet, previously shown to reduce red skeletal muscle insulin sensitivity by nearly 50% (38), we have been able to demonstrate chronic changes in cytosolic nPKC levels. Most importantly, use of BRL-49653, previously found to improve muscle insulin sensitivity in high-fat-fed and Zucker fa/fa rats (9, 28), partially reverses the defined chronic alterations in nPKCs. Because the drug also lowers plasma free fatty acid levels (28) and the DG content of muscle from fat-fed rats (27), the present work strengthens the argument for a role of PKC isoenzymes in mediating the decreased insulin sensitivity elicited by increased lipid availability, on stimulation of the kinases by lipid activators.
Although we have previously shown that assays of PKC activity are
broadly consistent with the results obtained by immunoblotting (34),
the assays are optimal for conventional PKC rather than nPKC activity
and are subject to artifacts, as discussed (34). In addition, PKC
assays are less informative than immunoblotting, which reveals the
behavior of individual isoenzymes, and we have therefore restricted the
PKC measurements in the present study to the latter. Our results
suggest that three nPKC isoenzymes could mediate inhibitory effects on
insulin signaling. nPKC appears
to be chronically activated without showing signs of downregulation, possibly in response to increased DG levels in muscle. In contrast, the
changes in nPKC
and
nPKC
were suggestive of both translocation and downregulation, although the decrease in cytosolic nPKC
was less marked than that
in nPKC
. This, together with
our previous observation that fat feeding also alters
nPKC
in white skeletal muscle
that is relatively insensitive to insulin (34), suggests that changes
in this isoenzyme may be less directly related to insulin sensitivity.
Our observations of the cytosolic depletion of
nPKC could be interpreted as an
indication that in fat-fed rats,
nPKC
is constantly
translocating to the membrane fraction, where it is subsequently
downregulated by proteolysis. A greater turnover of
nPKC
at the membrane might lead
to increased phosphorylation of protein substrates and thus inhibition
of insulin signaling, even though the rate of
nPKC
synthesis can no longer
maintain cytosolic nPKC
at
those levels seen in the starch-fed control rats. Although the
physiological functions of nPKC
have not been determined, it is interesting to note that this isoenzyme is poorly expressed in most tissues but is a major PKC in skeletal muscle (29).
Potential targets for PKC in disrupting insulin signaling have mainly
been identified in vitro and include the inhibition of the insulin
receptor tyrosine kinase by phosphorylation of the receptor -subunit
(6), inhibition of insulin-stimulated phosphorylation of IRS-1 (14),
inhibition of phosphatidylinositol 3-kinase activation (13) and
stimulation of tyrosine phosphatase activity (35). In addition PKC may
directly phosphorylate and inhibit glycogen synthase (1). However, the
precise mechanisms by which PKC isoenzymes act in the generation of
insulin resistance remain to be determined.
In some previous studies, acute insulin stimulation of tissues or cells
has led to translocation of PKC isoenzymes from soluble to membrane
fractions (4, 37, 40). Although the role of PKC in normal insulin
signaling is also uncertain (5, 20), recent evidence suggests that
atypical PKCs such as PKC could be involved in the insulin-mediated stimulation of
p21ras (18), gene transcription
(39), and glucose transport (4), whereas the translocation of
DG-sensitive isoenzymes could have inhibitory effects on glycogen
synthase activity (4). Our data are consistent with these
possibilities, as we observe translocation of DG-sensitive PKC
isoenzymes but not of atypical isoenzymes in insulin-resistant muscles.
We have previously described an acute redistribution of
PKC
in skeletal muscle during
euglycemic-hyperinsulinemic clamp (34). Because this translocation was
greater in fat-fed rats, compared with starch-fed controls, it may not
be a proximal signal but instead a secondary effect due to
insulin-stimulated changes in lipid flux. As glucose uptake into the
muscles from fat-fed rats was reduced (34), this again argued for an
inhibitory effect of PKC
on
insulin signaling. Acute effects of insulin on lipids and PKC
isoenzymes would make the interpretation of the effects of BRL-49653
treatment more complicated, and in the present study we have
concentrated on chronic PKC alterations in basal fat-fed rats.
A recent study of PKC isoenzyme expression in tissues of the type II
diabetic Goto-Kakizaki rat (2) found more widespread changes in
skeletal muscle PKC, relative to nondiabetic controls, than those seen
here. Translocation of PKC,
PKC
, and nPKC
were observed in addition
to that of nPKC
, whereas nPKC
levels were again
depleted. It was suggested that hyperinsulinemia played a role in the
elevation of DG and activation of PKC, because similar changes in PKC
isoenzymes were observed in normoglycemic, hyperinsulinemic obese aged
and obese Zucker rats (2). However, gross hyperinsulinemia is not
evident in the high-fat-fed rat (34), and we suggest that the more
specific changes observed here in nPKCs arise from increased lipid
availability to skeletal muscle. Consistent with that interpretation,
it should also be noted that BRL-49653 treatment reduces plasma insulin
levels in both starch- and fat-fed rats (28) but affects muscle PKC,
along with DG and insulin sensitivity, in fat-fed rats alone (Fig. 1, B-D,
and Refs. 27 and 28).
Another thiazolidinedione, troglitazone, has also been demonstrated to
affect membrane-associated nPKC
and nPKC
of ventricular
cardiomyocytes (3). However, in that study, a 30-min incubation with
the drug did not reduce translocation of PKC protein in response to the
DG analog phorbol 12-myristate 13-acetate (PMA) but indirectly caused
partial inhibition of the PKC activity that could be measured in
membrane fractions after PMA treatment. This acute effect of
troglitazone was accompanied by improvement of PMA-induced
desensitization of insulin-stimulated glucose transport (3), in
agreement with our findings that activated PKC is associated with
insulin resistance. Because acute treatment with troglitazone did not
affect PKC distribution (3), this argues against a direct effect of
thiazolidinediones on the kinases in muscle from fat-fed rats. Although
we did observe reduced translocation of nPKC isoenzymes in fat-fed rats
treated with BRL-49653, this difference may be explained by the chronic
nature of the treatment in our study (over 4 days) and suggests
different mechanisms both for the generation of insulin resistance by
PKC, activated either acutely by PMA or chronically by fat-feeding, and
for its amelioration in the shorter and longer term.
Thiazolidinediones have been found to improve several aspects of
insulin-mediated glucose metabolism, including glucose disposal rate,
in a large number of studies (see Ref. 33 for review). To date, the
only molecular target that has been identified for thiazolidinediones
is the peroxisome proliferator-activated receptor , which is
substantially expressed only in adipose tissue, and recent interest has
been focused on the stimulation of adipogenesis (33). Many of the
effects in muscle may therefore be related to altered adipose tissue
metabolism leading to reduced lipid availability to muscle. Our data
would support such a reduction by BRL-49653, leading to decreases in
muscle DG (or other lipid activators) and hence reduced PKC activation
and translocation. The proposed inhibitory effects of PKC on insulin
signaling would then be reduced, explaining the observed improvement in
muscle insulin sensitivity.
Diminished insulin action has been associated with elevated tissue levels of malonyl-CoA in the KKAy mouse (31) and in denervated muscle (32). This metabolite inhibits carnitine palmitoyltransferase 1, which catalyzes the rate-limiting step for long-chain fatty acyl-CoA (LCFA-CoA) transport into mitochondria, and it has been suggested that elevated cytosolic LCFA-CoAs play a central role in insulin resistance (30, 31). This could occur through various mechanisms, including activation of PKC (30, 31), either by direct stimulation (8) or via conversion to DG. Our results are consistent with this hypothesis, and it is likely that a high-fat diet leads to elevated cytosolic LCFA-CoAs in muscle through the increase in plasma free fatty acids and that this is reversed by treatment with BRL-49653 (28). Another thiazolidinedione, pioglitazone, lowered malonyl-CoA levels in both the KKAy mouse (31) and the Dahl salt-sensitive rat (24) and would also be expected to lower cytosolic LCFA-CoA and hence improve insulin action. Whether high-fat feeding also leads to elevated malonyl-CoA in muscle and whether this is reversed by BRL-49653 remain to be determined.
The direct activation of nPKCs by LCFA-CoAs or other lipids rather than
DG may explain our observation that
PKC was not altered by
fat-feeding, since this isoenzyme may be less sensitive to such
activation. Indeed, downregulation of cytosolic
nPKC
similar to that reported
here has been described in pancreatic
-cells incubated with
arachidonic acid, whereas PKC
was unaffected (22). However, as evidence for regulation of different DG species in distinct pools in skeletal muscle has also been reported
(10), an alternative explanation for the lack of diet effect on
PKC
is a possible separate
cellular localization of the isoenzyme and the elevated DG. Cellular
compartmentalization, as well as stimulation of nPKCs by as yet
undefined lipids, may also help to explain why, in contrast to our
findings with BRL-49653 in the fat-fed rat (27), muscle DG was in fact
elevated by pioglitazone in the
KKAy mouse (31), whereas insulin
action was improved in each case.
In conclusion, the data presented here are consistent with one or more PKC isoenzymes playing a causative role in the generation of fat-induced insulin resistance. Specific alterations in nPKCs are observed in response to high-fat feeding, previously shown to cause a reduction in skeletal muscle insulin sensitivity, and are partially reversed by treatment with an antidiabetic agent. Interpretation of these observations is not complicated by the presence of hyperglycemia or hyperinsulinemia in the fat-fed rats but again suggests that increased lipid availability is an important factor. To determine whether the affected nPKC isoenzymes do attenuate insulin signaling it will be necessary to identify substrates in muscle for these kinases, which might affect insulin action, and demonstrate that these undergo changes in phosphorylation state and activity in insulin-resistant states.
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ACKNOWLEDGEMENTS |
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This work was supported by a National Health and Medical Research Council Centre grant to the Garvan Institute of Medical Research and a Diabetes Australia research grant to T. J. Biden and C. Schmitz-Peiffer. The performance of animal studies was assisted by a grant from the Ramaciotti Foundations. BRL-49653 was kindly provided by SmithKline Beecham, Epsom, UK.
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FOOTNOTES |
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Current address of N. D. Oakes: Pharmacology CV, Astra Hässle AB, Mölndal, Sweden.
Address for reprint requests: C. Schmitz-Peiffer, Garvan Institute of Medical Research, 384 Victoria St., Darlinghurst, NSW 2010, Australia.
Received 23 May 1997; accepted in final form 23 July 1997.
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