Garvan Institute of Medical Research, Sydney, New South Wales 2010, Australia
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ABSTRACT |
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Muscle insulin
resistance in the chronic high-fat-fed rat is associated with increased
membrane translocation and activation of the novel, lipid-responsive,
protein kinase C (nPKC) isozymes PKC- and -
. Surprisingly,
fat-induced insulin resistance can be readily reversed by one
high-glucose low-fat meal, but the underlying mechanism is unclear.
Here, we have used this model to determine whether changes in the
translocation of PKC-
and -
are associated with the acute
reversal of insulin resistance. We measured cytosol and particulate
PKC-
and nPKC-
and -
in muscle in control chow-fed Wistar rats
(C) and 3-wk high-fat-fed rats with (HF-G) or without (HF-F) a single
high-glucose meal. PKC-
and -
were translocated to the membrane
in muscle of insulin-resistant HF-F rats. However, only membrane
PKC-
was reduced to the level of chow-fed controls when insulin
resistance was reversed in HF-G rats [% PKC-
at membrane,
23.0 ± 4.4% (C); 39.7 ± 3.4% (HF-F, P < 0.01 vs. C); 22.5 ± 2.7% (HF-G, P < 0.01 vs.
HF-F), by ANOVA]. We conclude that, although muscle localization of
both PKC-
and PKC-
are influenced by chronic dietary lipid
oversupply, PKC-
and PKC-
localization are differentially
influenced by acute withdrawal of dietary lipid. These results provide
further support for an association between PKC-
muscle cellular
localization and lipid-induced muscle insulin resistance and stress the
labile nature of high-fat diet-induced insulin resistance in the rat.
high-fat-fed rat; glucose; long-chain acyl-coenzyme A
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INTRODUCTION |
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INSULIN RESISTANCE is the major metabolic perturbation underlying non-insulin-dependent diabetes mellitus, obesity, and Syndrome X (24). Correlations between reduced insulin action and central adiposity in humans have led to the proposal that excess lipid availability contributes to the development of insulin resistance (5, 18). Furthermore, rats chronically fed a high-fat diet develop whole body and tissue-specific insulin resistance (25). A strong association in muscle between the accumulation of triglyceride and insulin resistance implicates local lipid availability as a putative regulator of muscle insulin sensitivity (21, 22, 26). Diacylglycerol and long-chain acyl-CoAs (LCA-CoAs), both of which are increased in the high-fat-fed rat, are activators of conventional and novel protein kinase Cs (nPKCs) (4, 20), and increases in PKC activity have been linked to reduced insulin action in humans and rats (2, 8, 23).
nPKC- and -
have been shown to translocate to the membrane in
skeletal muscle in response to chronic high-fat feeding in the rat
(23). Recently, it was reported (11) that
PKC-
moves to the membrane in rat skeletal muscle after a 5-h
infusion of lipid and that this translocation is associated with an
inhibition of insulin signaling. These observations suggest that nPKCs
may have an important role in the generation of insulin resistance. Indeed, PKCs have been shown to disrupt the insulin signal via serine
or threonine phosphorylation of the insulin receptor (3, 6,
27), insulin receptor substrate-1 (IRS-1) (9, 10) and potentially other proteins such as glycogen synthase
(1).
We previously demonstrated that a single high-glucose low-fat meal
readily reverses insulin resistance in the high-fat-fed rat
(21). This reversal appeared to be linked to a reduction of muscle triglyceride and LCA-CoA content that paralleled the reversal
of the muscle insulin resistance. In the present study, we have used
this model to further examine the link between insulin action and
skeletal muscle PKC total content and subcellular localization. We were
particularly interested in examining whether localization of individual
muscle PKC isozymes in the high-fat-fed rat was related to the chronic
increase in dietary lipid or tendency toward increased adiposity or to
acute muscle lipid metabolite availability and insulin sensitivity.
Differential responses were observed for the PKC isozymes PKC- and
-
.
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RESEARCH DESIGN AND METHODS |
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Materials.
Rabbit antipeptide antibodies against PKC- and nPKC-
were from
GIBCO BRL, Life Technologies (Mulgrave, Australia). Rabbit antipeptide
antibody against nPKC-
was purchased from Santa Cruz Biotechnology
(Santa Cruz, CA). Horseradish peroxidase-linked donkey anti-rabbit
antibody was from Jackson Immuno Research Laboratories (West Grove,
PA). Enhanced chemiluminescence reagents came from NEN (Boston, MA).
2-Deoxy-D-[2,6-3H]glucose
([3H]2-DG) and D-[U-14C]glucose
were from Amersham Pharmacia Biotech (Sydney, Australia). Male
Wistar rats were from the Animal Resource Centre (Perth, Australia).
Experimental animals and dietary treatment. All surgical and experimental procedures performed for this study were approved by the Animal Experimentation Ethics Committee (St. Vincents Hospital/Garvan Institute) and complied with the National Health and Medical Research Council of Australia Guidelines on Animal Experimentation.
Procedures were carried out as described previously (21). Male Wistar rats weighing ~250 g were fed a high-fat diet (350 kJ/day, at 1700) for 3 wk. The macronutrient composition of the fat diet, expressed as a percentage of total calories, was 59% fat, 20% carbohydrate, and 21% protein. On the day before assessment of insulin action, high-fat-fed rats were randomly split into two groups. One group was fed the normal high-fat meal (HF-F), whereas the second group was given a high-glucose, low-fat meal (HF-G) instead. The high-glucose meal was calorically equivalent to the high-fat meal but differed in the macronutrient composition: 10% fat, 69% carbohydrate, and 21% protein. To estimate the extent of the reversal, a third group of rats served as controls (C) and were fed chow (7% fat, 72% carbohydrate, and 21% protein) ad libitum. All rats underwent chronic jugular and carotid catheterization under ketamine hydrochloride (90 mg/kg)-xylazine (10 mg/kg) anesthesia 1 wk before clamp studies.Euglycemic clamp studies.
On the day of the clamp, food was removed at 0800, and studies were
performed between 1200 and 1500. Insulin action was assessed under the
conditions of the euglycemic-hyperinsulinemic glucose clamp as
previously described (16). Human insulin (Actrapid; Novo-Nordisk, Copenhagen, Denmark) was infused at a constant rate of
1.5 µmol · kg1 · h
1 (0.25 U · kg
1 · h
1), and blood
glucose was maintained at basal levels by a variable infusion of 30%
glucose. A tracer preparation of
D-[U-14C]glucose and 2-[3H]DG
was administered as an intravenous bolus ~75 min after commencement of the clamp for the determination of whole body glucose disposal (Rd)
and muscle glucose metabolic index (Rg').
Analytical methods. Plasma glucose and insulin levels were measured as previously described (17). Circulating leptin was determined with a leptin kit from Linco. Measurement of LCA-CoA in red quadriceps muscle was made using methods described previously with high-performance liquid chromatography (12). Results represent the sum of LCA-CoA species (C16:0, 18:1, 18:2).
Tissue extraction and immunoblotting.
Cytosolic and particulate fractions were prepared from red
gastrocnemius muscle by the method of Schmitz-Peiffer et al.
(23) and subjected to SDS-PAGE. Proteins were
electroblotted onto nitrocellulose membranes, which were then probed
with rabbit anti-peptide antibodies specific for PKC isozymes PKC-,
-
, and -
, followed by horseradish peroxidase-linked donkey
anti-rabbit antibody. PKC isozymes were visualized by enhanced
chemiluminescence and exposure to X-ray film. Densitometry of PKC bands
was performed with the use of a Medical Dynamics Personal Densitometer
SI and analyzed by IP Lab Gel H software (Signal Analytics, Vienna,
VA). Densities were corrected for variations in extraction between
samples using a dry weight (postextraction)-to-wet weight
(pre-extraction) ratio. PKC isozyme cytosolic and membrane levels were
determined relative to an internal PKC standard run in triplicate on
each gel. Total PKC levels represent the addition of cytosolic and
membrane fraction PKC levels. Data for PKC cellular distribution are
expressed as a percentage of the total.
Statistical analysis. All results are expressed as means ± SE. Analysis of data by ANOVA with appropriate post hoc tests (Fisher's protected least significant difference) was based on two a priori contrasts designed to examine differences between control and high-fat-fed (effect of fat diet) and between fat-fed and glucose groups (effect of reversal by a single high-glucose meal). Statistical calculations were performed using Statview SE+ Graphics for Macintosh (Abacus Concepts, Berkeley, CA). Differences were considered statistically significant at P < 0.05.
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RESULTS |
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Animal characteristics.
Body weights, fat pad weights (epididymal and inguinal,
expressed as % total body weight), and plasma parameters are shown in
Table 1. Rats fed a high-fat diet for 3 wk (HF-F, HF-G) had a significantly greater fat pad mass than
control chow-fed rats (HF-F 7.8 ± 0.3 g and HF-G 8.0 ± 0.3 g vs. C 6.4 ± 0.4 g, P < 0.01).
Circulating leptin levels reflected the increase in body adiposity.
These parameters were not altered by a single high-glucose meal,
indicating that the mechanism of the reversal of insulin resistance was
not attributable to a reduction in body fat or change in plasma leptin
levels. Basal glucose levels were not different among groups; however,
basal insulin levels were markedly reduced in the HF-G group compared
with HF-F, suggesting an improvement in whole body insulin sensitivity.
Plasma glucose and insulin concentrations were equivalent in all groups
under euglycemic-hyperinsulinemic clamp conditions.
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Effect of diet on LCA-CoA species (C16:0, 18:1, 18:2) in red
skeletal muscle.
The level of muscle LCA-CoAs was significantly increased in the HF-F
rats (Fig. 1) despite the level
of variability observed in this group. Hyperinsulinemia during the
euglycemic clamp tended to decrease the LCA-CoA content of muscle from
control and both HF groups, but this change was not significant. It
should be noted that the level of LCA-CoA in the clamped HF group was
still significantly higher than the level observed in control muscle in
either the basal or the clamped state. In the muscle from HF-G rats,
the clamp procedure led to a significant decrease in LCA-CoA content to
levels similar to that found in control muscle.
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Effect of diet on total PKC levels and subcellular distribution of
PKC in red gastrocnemius muscle.
Dietary manipulations did not alter the total muscle levels of PKC-
and -
(Table 3). Total levels of
PKC-
were significantly downregulated in the HF-F and HF-G groups
compared with control.
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DISCUSSION |
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Restricting dietary lipid or replacing calories from lipid with glucose produces alterations in the metabolic responses of the chronically high-fat-fed rat within 15 h. Insulin sensitivity as estimated by the clamp GIR and glucose disposal as estimated by the whole body (Rd) and/or skeletal muscle (Rg'; Table 2) are significantly improved and are associated with a reduction in basal insulin levels. Reversal of dietary-induced insulin resistance by a single high-glucose low-fat meal is a unique observation and has been examined to some extent in our laboratory (21).
In this study, we have shown that skeletal muscle PKC- is increased
at the membrane with high-fat feeding but is rapidly redistributed with
the acute reversal of fat-induced insulin resistance by a single
low-fat high-glucose meal under clamp conditions. In addition, the
amount of PKC-
at the membrane correlated significantly with muscle
insulin-stimulated glucose uptake in all groups, suggesting a
relationship between the subcellular localization of PKC-
and insulin action. There was also a significant decrease in the total amount of PKC-
in both the HF-F and HF-G rats, implying a chronic activation and downregulation of PKC-
with high-fat feeding.
The present results suggest that PKC- and -
are different in
their responses to altered dietary intake. The distribution of PKC-
is, like PKC-
, increased in the membrane fraction with chronic
high-fat feeding. However, the reversal of insulin resistance was not
associated with subsequent alterations in PKC-
subcellular localization. PKC-
may, of course, still play a role in the
generation of insulin resistance.
Reduced phosphatidylinositol (PI) 3-kinase activation and GLUT-4 translocation accompany insulin resistance induced by high-fat feeding in rodents (28). Although direct measurement of insulin signaling factors was not carried out in the present study, there is considerable evidence that activation of PKC can inhibit the signaling process. PKCs may mediate serine/threonine phosphorylation of the insulin receptor (3, 13, 19, 27) and also of IRS-1 (10, 7, 10, 14), leading to diminished IRS-1 tyrosine phosphorylation and, hence, downstream signaling.
High-fat-fed rats have increased muscle lipid intermediates (21,
23), such as diacylglycerol and LCA-CoAs, which activate nPKCs
(4, 20). Thus activation of nPKCs in vivo provides a
possible mechanism for lipid-induced insulin resistance. Further evidence for nPKC involvement in mediating insulin sensitivity has
arisen from a study in which treatment with the thiazolidinedione BRL-49653 improved insulin action in the high-fat-fed rat and partially
reversed the translocation of nPKC-, -
, and -
(24). Furthermore, in muscle from 5-h Liposyn-infused
insulin-resistant rats, PKC-
is increased at the membrane, IRS-1
tyrosine phosphorylation is reduced, and PI 3-kinase activity is
inhibited (11). As shown here in the high-fat-fed rat
given a single high-glucose meal, where dietary lipid is acutely
withdrawn, muscle LCA-CoAs are reduced to control levels upon insulin
stimulation, and this change parallels the reversal of insulin
resistance and the cellular redistribution of PKC-
observed in this study.
In the present study, adiposity in the high-fat-fed rat, as determined by fat pad weights and circulating leptin, was unaltered with the reversal of insulin resistance. These findings suggest that muscle lipid supply, rather than whole body lipidemia, reflects short-term dietary fat intake in the rat, because the mobilization and metabolism of muscle lipid metabolites appear to be acutely attenuated by dietary lipid withdrawal.
Muscle insulin resistance is not observed after 3 days of high-fat feeding in rats and requires a longer dietary period to develop (15). Therefore, it is unlikely that an acute manipulation such as a reduction in dietary lipid for one meal would result in a full reversal of the fat diet-induced whole body insulin resistance; rather, it would seem that a compensatory mechanism is rapidly turned on to increase insulin-stimulated muscle glucose uptake.
Although the mechanism responsible for the reversal in muscle insulin
resistance in high-fat-fed rats remains to be elucidated, it is
possible that it is the reduced lipid availability to the muscle and
subsequent redistribution of PKC- that alleviates an inhibition on
insulin signaling. Such a mechanism could involve a decrease in
metabolically active lipids such as LCA-CoAs, as was found, resulting
in a reduction in the activation of PKC-
. It is hypothesized that,
in skeletal muscle, PKC-
inhibits the insulin signaling pathway in
high-fat-induced insulin resistance in the rat. Thus a reduction in
active PKC-
would improve signal transduction and, consequently,
insulin action. Conversely, the lowering of LCA-CoAs and PKC-
at the
membrane may be a consequence of the improvement in muscle insulin
action, because both of these parameters were not changed in
high-glucose-fed rats before insulin stimulation.
Ideally, direct measurement of specific PKC isozyme activity rather than comparison of PKC distribution into cytosolic and particulate fractions in skeletal muscle would provide stronger evidence for their chronic activation in fat-induced muscle insulin resistance and inactivation after a single high-glucose meal. However, specific substrates for different PKC isozymes have yet to be identified, and changes in general PKC activity cannot determine the importance of different isozymes in metabolic processes. In addition, measurement of the activity of different intermediates in the insulin signaling pathway is critical for the elucidation of a mechanism, and this is the subject of further investigations.
The results of this study show that PKC- localization can be acutely
regulated in association with alterations in insulin-stimulated glucose
uptake. Both muscle insulin action and PKC-
localization are very
labile in the high-fat-fed rat, and they appear very dependent on
recent dietary fat intake and less dependent on whole body adiposity as
indicated by percent body fat. Because the amount of PKC-
at the
membrane is rapidly decreased with the reversal of fat-induced insulin
resistance, this relationship between PKC-
activation and insulin
action in skeletal muscle may be important in our understanding of
insulin resistance.
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ACKNOWLEDGEMENTS |
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We are grateful for the expert support of our animal research program, Dr. Julie Ferguson and staff, and the technical assistance of Joanna Edema.
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FOOTNOTES |
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This study was supported by funding from the National Health and Medical Research Council of Australia.
Address for reprint requests and other correspondence: K. S. Bell, Garvan Institute of Medical Research, 384 Victoria St., Darlinghurst, NSW 2010, Australia.
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. Section 1734 solely to indicate this fact.
Received 26 January 2000; accepted in final form 6 July 2000.
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