Departments of 1 Physiology and 2 Medicine, University of Toronto, Toronto M5S 1A8, Canada
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ABSTRACT |
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The
mechanisms of the impairment in hepatic glucose metabolism
induced by free fatty acids (FFAs) and the importance of FFA oxidation
in these mechanisms remain unclear. FFA-induced peripheral insulin
resistance has been linked to membrane translocation of novel protein
kinase C (PKC) isoforms, but the role of PKC in hepatic insulin
resistance has not been assessed. To investigate the biochemical
pathways that are induced by FFA in the liver and their relation to
glucose metabolism in vivo, we determined endogenous glucose production
(EGP), the hepatic content of citrate (product of acetyl-CoA derived
from FFA oxidation and oxaloacetate), and hepatic PKC isoform
translocation after 2 and 7 h Intralipid + heparin (IH) or
SAL in rats. Experiments were performed in the basal state and during
hyperinsulinemic clamps (insulin infusion rate, 5 mU · kg1 · min
1). IH
increased EGP in the basal state (P < 0.001) and
during hyperinsulinemia (P < 0.001) at 2 and 7 h.
Also, 7-h infusion of IH induced resistance to the suppressive effect
of insulin on EGP (P < 0.05). Glycerol infusion
(resulting in plasma glycerol levels similar to IH infusion) did not
have any effect on EGP. IH increased hepatic citrate content by
twofold, independent of the insulin levels and the duration of IH
infusion. IH induced hepatic PKC-
translocation from the cytosolic
to membrane fraction in all groups. PKC-
translocation was greater
at 7 compared with 2 h (P < 0.05). In conclusion,
1) increased FFA oxidation may contribute to the FFA-induced
increase in EGP in the basal state and during hyperinsulinemia but is
not associated with FFA-induced hepatic insulin resistance, and
2) the progressive insulin resistance induced by FFA in the
liver is associated with a progressive increase in hepatic PKC-
translocation.
hepatic glucose production; free fatty acid oxidation; hyperinsulinemic-euglycemic clamp
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INTRODUCTION |
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THE ASSOCIATION between obesity, insulin resistance, and type 2 diabetes mellitus is well documented (for review, see Refs. 4, 22, 25, 28, and 32). Free fatty acids (FFA) have been implicated as an important causative link in this association. An elevation of plasma FFA has been shown to impair insulin action and to be a risk factor for the development of type 2 diabetes (38). A number of groups have investigated the mechanisms that underlie the FFA-induced impairment of glucose metabolism in muscle (5, 15, 19, 20, 23, 37, 39, 43, 46), but little is known about the mechanisms of the FFA-induced impairment of glucose metabolism in the liver (30, 37).
In the liver, FFAs increase gluconeogenesis both in vitro and in vivo (4, 11, 54), and a large number of studies have demonstrated that Intralipid + heparin (IH) increases endogenous glucose production (EGP) during euglycemic clamps (3, 5, 29, 42, 45, 47, 51). However, the time course of this increase in EGP is not known. Intralipid has a high content of free glycerol, and both glycerol and FFA are released from the triglycerides of Intralipid. In most studies, because glycerol was not infused as a control, it cannot be excluded that the effect of IH on EGP was due, at least in part, to the effect of glycerol, as glycerol is a gluconeogenic precursor.
Randle et al. (41) have shown that FFAs compete with glucose for substrate oxidation (this has been termed the glucose-fatty acid cycle) in isolated rat heart muscle and diaphragm. With regard to the mechanism of the FFA effect on hepatic glucose metabolism, a Randle-like mechanism has been invoked to explain the FFA-induced stimulation of gluconeogenesis, i.e., acetyl-CoA derived from FFA oxidation allosterically activates pyruvate carboxylase, and NADH, also a product of FFA oxidation, is used for the formation of glyceraldehyde 3-phosphate from 1,3-bisphosphoglycerate. Additionally, citrate-induced inhibition of phosphofructokinase-1 was observed in the perfused rat liver and in isolated hepatocytes exposed to FFA (31, 35, 52). However, the finding that hepatic insulin resistance in high-fat-fed rats was not ameliorated by etomoxir (37), an inhibitor of fatty acid oxidation, suggests that other mechanisms are likely involved. FFA-induced protein kinase C (PKC) activation has been investigated as a potential mechanism responsible for the FFA-induced insulin resistance in muscle (19); however, the role of PKC in FFA-induced hepatic insulin resistance is not known. It has been observed that in hepatocytes, oleic acid promotes translocation of PKC from the cytosol to the plasma membrane (14).
The present study was performed 1) to examine the time course of the effect of FFA on EGP and 2) to determine whether the postulated FFA-induced increase in EGP is associated with evidence of increased FFA oxidation and/or PKC activation.
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RESEARCH DESIGN AND METHODS |
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Animal models. Normal female Wistar rats (Charles River, St-Constant, QC, Canada) weighing 250-300 g were used for experiments. Female Wistar rats were used to allow for future comparison of the effects of FFA with those on female Zucker Diabetic Fatty rats. The latter are a convenient model of high-fat diet-induced diabetes (13). The rats were housed in the Univ. of Toronto Dept. of Comparative Medicine. They were exposed to a 12:12-h light-dark cycle and were fed rat chow (Purina no. 5001, 4.5% fat; Ralston Purina, St. Louis, MO) and water ad libitum. The Animal Care Committee of the Univ. of Toronto approved all procedures.
Surgical procedures. After 3-5 days of adaptation to the facility, rats were anesthetized with ketamine-xylazine-acepromazine (100:0.1:0.5 mg/ml, 1 µl/g body wt), and indwelling catheters were inserted into the right internal jugular vein for infusions and the left carotid artery for sampling. Polyethylene catheters (PE-50; Clay Adams, Becton Dickinson, Sparks, MD), each extended with a segment of Silastic tubing (length of 3 cm, internal diameter of 0.02 in.; Dow Corning, Midland, MI), were used. The venous catheter was extended to the level of the right atrium, and the arterial catheter was advanced to the level of the aortic arch. Both catheters were tunneled subcutaneously and exteriorized. The catheters were filled with a mixture of 60% polyvinylpyrrolidone and heparin (1,000 U/ml) to maintain patency and were closed at the end with a metal pin. The rats were allowed a minimum 3- to 4-day period of postsurgery recovery before experiments.
Experimental design. The rats were fasted overnight and randomized to two groups, one of which received IH infusion (20% Intralipid + 20 U/ml heparin, 5.5 µl/min), while the other group was a saline (SAL; equivolume)-treated control. Both IH- and SAL-treated rats were randomly assigned to different protocols that varied in the duration of IH/SAL infusion and in the conditions of the experimental determinations. The duration of IH and SAL was 2 and 7 h, and experimental determinations were made in the basal fasting state and during hyperinsulinemic-euglycemic clamp.
For the 2-h IH basal and the 2-h SAL basal protocols, IH or SAL was infused intravenously for 2 h through the jugular catheter together with [6-3H]glucose (20 µCi, bolus + 0.4 µCi/min infusion) to assess the metabolic clearance rate (MCR) of glucose and EGP. The 2-h SAL clamp and the 2-h IH clamp protocols were similar to the 2-h SAL basal and the 2-h IH basal protocols, with the addition of an intravenous infusion of insulin (5 mU · kgLaboratory methods. Plasma glucose was measured with a Beckman Glucose Analyzer II (Beckman, Fullerton, CA). Plasma radioactivity from [6-3H]glucose was determined after deproteinization with Ba(OH)2 and ZnSO4, passage through ion exchange columns, and subsequent evaporation. Aliquots of the [6-3H]glucose and of the tritiated glucose infusate were assayed together with the plasma samples. The intra-assay coefficient of variation was 2.5%, and the interassay coefficient of variation was 6.5%. Insulin and C-peptide levels in plasma were determined by radioimmunoassays (RIAs) by using kits specific for rat insulin (but with 100% cross-reactivity with porcine insulin used for infusion) and C-peptide from Linco Research (St. Charles, MO). The coefficients of variation were <9 and 10.5% for insulin and C-peptide, respectively. Plasma FFA levels were measured using a colorimetric kit from Wako Industrials (Osaka, Japan). Plasma triglyceride levels and glycerol levels were also measured using colorimetric kits from Boehringer Mannheim (Mannheim, Germany).
Hepatic content of citrate.
Hepatic content of citrate was measured as an indicator of FFA
oxidation in the presence of a source of pyruvate (precursor of
oxaloacetate). Citrate is the product of acetyl-CoA (derived from
-oxidation of fatty acids) and oxaloacetate, the production of which
from pyruvate is increased by the allosteric effect of acetyl-CoA on
pyruvate carboxylase. Citrate accumulates when Krebs cycle enzyme
activity is slowed down by NADH (from substrate oxidation). For citrate
assay, liver samples were snap-frozen in liquid nitrogen at the end of
the experiment and stored at
70°C. The samples were homogenized
with 13% perchloric acid, precipitated proteins were removed by
centrifugation, and the supernatants were neutralized by adding 2 N
KH2CO3. The precipitated potassium perchlorate
salt was eliminated by centrifugation, and the resulting supernatants were stored at
20°C and subsequently used for citrate measurements. Citrate was assayed by coupling the citrate lyase/malate dehydrogenase reactions according to Williamson and Corkey (53). Total
cellular proteins were measured by the Bradford assay from BioRad
(Mississauga, ON, Canada) after dissolution of the trichloroacetic
acid-precipitated protein pellets in 1 N NaOH.
PKC isoform translocation.
The translocation of the 2,3-diacylglycerol (DAG)-sensitive
isoforms of PKC from cytosol to membrane reflects their activation and
was assessed by comparing immunoblots of the cytosolic and membrane-associated fractions. Initial studies were performed in which
we examined the expression of the PKC isoforms-, -
, -
, -
,
and -
using the following antibodies: polyclonal antibody specific
for PKC-
, -
, -
, and -
from Sigma and -
from Santa Cruz
Biotechnology (Santa Cruz, CA). All isoforms were detected in the rat
liver. In preliminary experiments, we found that only PKC-
showed
evidence of membrane translocation induced by IH. Therefore, we
concentrated our studies on PKC-
. Liver samples (150 mg) were
homogenized by a hand-held glass homogenizer in buffer A (50 mM Tris · HCl, pH 7.5; 10 mM EGTA; 2 mM EDTA; 1 mM NaHCO3; 5 mM MgCl2; 1 mM
Na3VO4; 1 mM NaF; 1 µg/ml aprotinin, leupeptin, and pepstatin; 0.1 mM phenylmethylsulfonyl fluoride; and 1 µM microcystin). The homogenates were centrifuged at 100,000 g for 1 h at 4°C, and the supernatants were retained
as the cytosolic fraction. The pellet was resuspended in buffer
B (buffer A + 1% Triton X-100), homogenized by
passing through a 23-gauge needle three times, incubated for 15 min on
ice, and centrifuged at 100,000 g for 1 h at 4°C. The
supernatant provided the solubilized membrane fraction. The purity of
the cytosolic and membrane fractions was assessed by assaying
glucose-6-phosphate dehydrogenase (Sigma) and 5'-nucleotidase
activities (Sigma), respectively. The results showed that the index of
purity of both fractions was >90%. The protein concentration in all
samples was determined by the detergent-compatible modified Lowry
microassay (BioRad), using serum albumin as the standard. Fifty
micrograms of protein in all samples were mixed with equal volumes of
3× sample-loading buffer (6.86 M urea, 4.29% SDS, 300 mM
dithiothreitol, and 43 mM Tris · HCl, pH 6.8) and left at room
temperature for 30 min. The mixture was then vortexed and subjected to
SDS-PAGE (10% polyacrylamide). After electrophoretic separation,
proteins were transferred to Immobilon-P membranes. The membranes were
then incubated for 1 h at 4°C in Tris-buffered saline-Tween
(TBST) containing 5% nonfat dried milk, pH 7.4. After the blocking
step, membranes were washed in rinsing solution (TBST, pH 7.4) and then
incubated overnight at a concentration of 1:1,000 with an
affinity-purified polyclonal antibody specific for PKC-
(Sigma).
After washing, membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (Amersham, Baie d'Urfe, Quebec). The membranes were then washed several times with TBST and
developed with the use of enhanced chemiluminescence (ECL; Zymed
Laboratories, San Francisco, CA). The bands obtained from immunoblotting were quantified by scanning laser densitometry.
Calculations. Glucose turnover (rate of appearance of glucose determined with [6-3H]glucose) was calculated using steady-state formulas (49), taking into account the extra tracer infused with the glucose infusate (17). In the basal state, the total rate of glucose appearance corresponds to the EGP. During the clamps, EGP was calculated by subtracting the exogenous glucose infusion rate from the total rate of glucose appearance. At steady state, glucose disappearance corresponds to the rate of glucose appearance, and at euglycemia, glucose disappearance corresponds to tissue glucose utilization, because renal glucose clearance is zero. MCR of glucose is defined as glucose utilization divided by the plasma glucose levels. Data are presented as average values of the samples that were taken in the last 30 min of the experiment.
Statistical analysis. One-way analysis of variance for repeated measures was used to compare differences between treatments (SAL vs. IH). Two-way analysis of variance with interaction was used to compare differences between the effects of IH at 2 and 7 h, using treatment (SAL vs. IH) and duration of infusion (2 vs. 7 h) as independent variables in both the basal and the clamp groups. Two-way analysis of variance with interaction was used to compare differences between the effects of IH in basal and clamp groups, using treatment (SAL vs. IH) and experimental conditions (basal vs. clamp) as independent variables at both 2 and 7 h. Statistical calculations were performed using SAS software (Statistical Analysis System, Cary, NC). Significance was accepted at P < 0.05.
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RESULTS |
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IH elevated plasma FFA levels by approximately three- to fourfold
in all groups (P < 0.001; Table
1), and the levels of FFAs were lower
during the hyperinsulinemic clamps than during the basal fasting state,
as expected. The triglyceride and glycerol levels were also elevated by
IH and were lower during the clamps than during the basal experiments
(Table 1). Plasma glucose levels were higher with IH vs. SAL infusion
in the basal experiments but were maintained at ~6.5 mM during the
hyperinsulinemic clamps (Table 1). IH significantly
increased plasma C-peptide levels in the basal experiments (Table 1),
suggesting that FFAs increased endogenous insulin secretion. During the
clamps, C-peptide levels were very low, and IH infusion did not
increase C-peptide levels (Table 1), indicating that insulin secretion
was almost completely suppressed by exogenous hyperinsulinemia with
both SAL and IH infusion.
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IH significantly increased plasma insulin levels in the basal
experiments (Fig. 1), consistent with the
increase in C-peptide. Of interest, insulin levels were also higher
with IH in the clamp experiments despite the suppressed C-peptide,
consistent with a decrease in insulin clearance as we have previously
shown (51). This effect of IH was greater in the 7-h IH
clamp vs. 2-h IH clamp group (P < 0.001; Fig. 1). When
exogenous insulin infusion was given at one-half the original rate (2.5 mU · kg1 · min
1) in the 7-h
IH clamp (1/2 Ins) group, the plasma insulin levels were matched with
those observed in the 2-h IH clamp or 7-h SAL clamp group.
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IH significantly decreased glucose infusion rate (Ginf) at 2 and 7 h (Table 2). The effect of IH vs. the
corresponding SAL experiments was greater at 7 vs. 2 h
(P < 0.001; Table 2), indicating that IH induced whole
body insulin resistance in a time-dependent fashion. In basal
steady-state conditions, the rate of appearance of glucose (EGP) is
equal to the rate of disappearance of glucose (glucose utilization;
GU). IH was found to increase basal EGP = GU after 2 and 7 h
of infusion (P < 0.001; Table 2). During the clamps,
IH decreased GU after 2 h, and the effect of IH vs. the
corresponding SAL experiments was significantly greater
(P < 0.01) after 7-h infusions (Table 2). Glucose MCR
is equal to GU divided by plasma glucose. IH did not have significant
effects on glucose MCR in the basal state, presumably because
of the increased endogenous insulin levels (Fig.
2). IH decreased the insulin
stimulation of glucose MCR during the clamps vs. the corresponding SAL
experiments, and the effect was greater after 7 h than after
2 h (P < 0.001), indicating a time-dependent
augmentation of FFA-induced peripheral insulin resistance (Fig. 2).
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IH increased EGP = GU in the basal fasting state at both 2 and
7 h as reported above (Table 2 and Fig.
3). There was a tendency for the effect
of IH on basal EGP to be greater after 7 h than after 2 h
(P = 0.07). During the hyperinsulinemic clamps, IH
increased EGP at 2 and 7 h (Fig. 3; 2-h SAL = 21 ± 3, 2-h IH = 46 ± 5, 7-h SAL = 21 ± 5, 7-h IH = 48 ± 5 µmol/kg1 · min
1; SAL
vs. IH, P < 0.001). To assess the effect of insulin on
EGP, the suppression of EGP between basal and clamp conditions was compared in the SAL and IH protocols. IH tended to decrease the ability
of insulin to suppress EGP at 2 h from ~50 to 30% and at 7 h from 54 to 28%. These impairments did not reach statistical significance but were observed despite the higher levels of circulating insulin in the IH group. When the insulin levels were matched between
7-h SAL and 7-h IH clamp (1/2 Ins), IH markedly decreased the ability
of insulin to suppress EGP from ~54 to ~6% (P < 0.05; Fig. 3), indicating hepatic insulin resistance. Furthermore, at matched insulin levels between 7-h IH clamp (1/2 Ins) and 2-h IH clamp,
the insulin-induced suppression of EGP was markedly lower with 7-h than
2-h IH infusion, indicating that hepatic insulin resistance was
progressive over time.
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To exclude possible effects of glycerol on EGP during IH infusion,
glycerol was infused for 7 h (5 mg · kg1 · min
1), resulting
in plasma glycerol levels similar to 7-h IH in the basal state (Table
3) and even higher than that observed in
7-h IH during the clamps (Table 3). The glycerol infusion had no effect
on EGP compared with SAL infusion either in the basal fasting state or
during the hyperinsulinemic-euglycemic clamp (Table 3). These findings
suggest that the effects of IH on EGP are due largely, if not entirely,
to FFAs rather than glycerol.
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To investigate the mechanisms responsible for FFA-induced impairment in
hepatic glucose metabolism, we measured citrate content (to indirectly
assess FFA oxidation) as well as PKC -isoform translocation. IH
increased citrate content by approximately twofold (P < 0.05) in all groups (Table 4), and the
elevation was independent of the insulin levels and the duration of IH
infusion, being the same in the basal and clamp experiments and at 2 and 7 h. IH induced hepatic PKC-
translocation from the
cytosolic to the membrane fraction in all groups (Figs.
4 and
5). The degree of PKC-
translocation was not different between basal and clamp conditions
(Figs. 4 and 5). Thus the data from the basal and clamp studies were
combined, and PKC-
translocation induced by IH was found to be
greater at 7 h (membrane-to-cytosolic ratio: 4.22 ± 0.22)
vs. 2 h (membrane-to-cytosolic ratio: 3.01 ± 0.16)
(P < 0.05). Hepatic PKC-
translocation and citrate
content were not measured in the IH clamp experiments with exogenous
insulin infused at 2.5 mU · kg
1 · min
1 because
their levels were not found to be dependent on insulin levels. As
stated in RESEARCH DESIGN AND METHODS, we focused on PKC-
because we found no consistent translocation of hepatic PKC-
, -
, -
, and -
in response to IH in preliminary studies. Figure 6 shows some of these preliminary
studies indicating that 7-h IH infusion did not induce hepatic PKC-
or PKC-
translocation.
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DISCUSSION |
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In this study, the effects of FFA on hepatic glucose metabolism
and the biochemical mechanisms that underlie these effects were
examined. IH elevated basal plasma FFA to levels that were above the
physiological range but within the FFA elevation seen in uncontrolled
diabetes. The FFA levels in the clamps were lower than the basal FFA
levels, which is consistent with the antilipolytic and FFA
reesterification effects of insulin (7). IH increased plasma insulin levels in all groups because of increased insulin secretion in the basal state and a decreased insulin clearance during
the clamp. The FFA-induced impairment in insulin clearance has been
demonstrated in previous in vivo studies (8, 21, 34, 51).
The mechanism for this impairment is likely a decrease in hepatocyte
insulin binding, which our in vitro studies suggest is caused in part
by FFA-induced PKC activation (10). In the present study,
there was a progressive impairment of insulin clearance over time,
which, interestingly, paralleled the increase in hepatic PKC- translocation.
It is well established that FFAs induce peripheral insulin resistance
by an inhibition of insulin-stimulated glucose uptake in skeletal
muscle. During the clamps, IH decreased GU and MCR in a time-dependent
fashion, indicating marked insulin resistance. This time-dependent
augmentation of peripheral insulin resistance is consistent with
previous findings (20, 43). The suggested mechanism is a
decrease in insulin receptor substrate-1 (IRS-1)-associated phosphatidylinositol 3-kinase activity, which is mediated by PKC- activation in muscle (15, 19).
In the basal state, IH increased EGP after 2- and 7-h infusions despite increased insulin and glucose concentrations. Previous studies in humans revealed that IH increased gluconeogenesis but did not have any effects on EGP under basal conditions (6, 44). However, when endogenous insulin secretion was inhibited by somatostatin and exogenous insulin was given to maintain insulin at basal levels, IH increased EGP in some studies (6) but not in others (44). IH was also found to increase EGP during insulinopenia achieved by somatostatin infusion without insulin replacement (1, 16). Taken together, these studies suggest that in humans, the stimulatory effect of IH on EGP in the basal state is counteracted by an increase in insulin secretion. Furthermore, there may be autoregulation of basal EGP independent of an increase in basal insulin (44).
In 5-h-fasted rats, the lowering of plasma FFA levels with the use of acipimox did not affect basal EGP (27). However, it has recently been demonstrated by Bergeron et al. (2) that in 12-h-fasted rats, a fourfold elevation of FFAs achieved by IH infusion increases basal EGP (although not significantly) by ~25% despite an increase in plasma insulin concentration. Also, Song et al. (48) have recently shown that in overnight-fasted rats, high-fat diet increases basal EGP in the presence of elevated plasma insulin levels. The difference between the 5-h-fasted and overnight-fasted rats may be due to the fact that after overnight fasting, glycogenolysis is limited by glycogen depletion (27, 48) and may not further decrease to provide autoregulation of basal EGP in the presence of FFA-stimulated gluconeogenesis. Thus the latter would lead to a FFA-induced increase in basal EGP.
In our studies (overnight-fasted rats), an increase in FFA oxidation
(as indicated by an increase in hepatic citrate content) may be partly
responsible for the increase in basal EGP. Our preliminary data suggest
that an allosteric stimulatory effect of fatty acyl-CoA on
glucose-6-phosphatase also could have contributed to the increase in
basal EGP (Lam TKT, van de Werve G, and Giacca A, unpublished observations). This effect could have further impaired
autoregulation of basal EGP in the presence of an increase in
gluconeogenesis induced by FFA oxidation. In addition, some degree of
hepatic insulin resistance, as suggested by increased PKC-
translocation, may have contributed to the increase in basal EGP by
decreasing the ability of insulin to suppress glycogenolysis. Hepatic
insulin resistance, measured by a reduced ability of insulin to
suppress EGP during the clamps, was evident after 7 h of IH
infusion. After 2 h of IH infusion, the decrease of the insulin
effect on EGP was not significant; however, it occurred in the presence
of a slight elevation in insulin levels, which suggests some impairment in insulin action.
In our animal model, 5 mU · kg1 · min
1 of insulin
infusion (resulting in ~ 600 pM of plasma insulin) suppressed
EGP by ~55%. This is in accordance with previous studies conducted
at similar plasma insulin levels by Giaccari et al. (18)
and Miles et al. (33). During the hyperinsulinemic clamps,
IH increased EGP in both 2- and 7-h experiments and induced marked
hepatic insulin resistance at 7 h, as described in
RESULTS. The increase in EGP induced by IH during
hyperinsulinemia is in accordance with previous studies where the
effect of IH was compared with saline (3, 5, 29, 42, 45, 47,
51). It is also in accordance with studies that compared the
effects of IH and glycerol infusions in dogs and humans (5,
42). This increase has been variably interpreted as a lack of
autoregulation, as glycogenolysis is already suppressed by
hyperinsulinemia, or as specific impairment of hepatic insulin action
(28).
In our studies, the increase in FFA oxidation (indicated by an increase in hepatic citrate content) may have contributed partly to the IH-induced increase in EGP under hyperinsulinemic clamp conditions. However, an increase in FFA oxidation does not appear to explain the progressive decrease of insulin's ability to suppress EGP induced by IH infusion, since the change in hepatic citrate content was independent of the duration of IH infusion. This suggests that, similar to peripheral tissues, other mechanisms are responsible for the FFA-induced insulin resistance in the liver.
High-fat feeding has been shown to induce skeletal muscle insulin
resistance in association with membrane translocation of PKC- and
-
(46), and, similarly, lipid infusion has been
associated with membrane translocation of PKC-
(19).
Both PKC-
and -
are novel isoforms of PKC. In the present study,
IH was found to induce the translocation of hepatic PKC-
(also a
novel PKC) from the cytosolic to the membrane fraction in all groups.
This suggested that PKC-
translocation could also have contributed to the increase in EGP induced by IH both in the basal state (at least
at 7 h, when insulin resistance was observed) and during the
clamps. Notably, the effect of PKC-
translocation induced by IH was
significantly greater at 7 vs. 2 h, which may be due to a
progressive accumulation of long-chain fatty acyl-CoA and diacylglycerol activating this isoform (note that FFA oxidation did not
appear to increase over time). In addition, the membrane-to-cytosol ratio of PKC-
was similar in the basal state and the
hyperinsulinemic clamps, indicating that in our model of 2-h continuous
insulin infusion, contrary to other models (9), insulin
does not activate this isoform of PKC in the liver. Thus PKC-
translocation increased over time, independent of insulin levels. Under
conditions of hyperinsulinemia, the progressive increase of PKC-
translocation was associated with a progressive increase in EGP,
suggesting a specific PKC-mediated impairment in hepatic insulin
action, which was obviously more evident at high than at basal insulin levels.
Previous studies have reported that in obese hypertriglyceridemic
diabetic rats, hepatic PKC activity is greater than in lean rats
(40). Considine et al. (12) have shown that
obese diabetic rats and obese subjects with type 2 diabetes have higher
hepatic membrane-associated PKC-, -
, and -
than controls.
Normalization of circulating glucose levels in obese diabetic rats did
not result in reduction of hepatic membrane PKC content, suggesting
that factors other than hyperglycemia were responsible for this
finding. These factors may include elevated FFA and triglyceride
levels, which were not measured in that study (12). In the
same study, PKC-
was not detected in human liver (using a different
antibody from that used in our study) and was not assayed in rats.
However, PKC-
was immunodetected in a recent study in normal human
liver with the use of a different antibody from ours (50)
and from that used in the study of Considine et al. Also, PKC-
mRNA
was found to be expressed in human liver in another study
(26). In the human hepatoma cell line HepG2, PKC-
and
-
membrane translocation was found to mediate the downregulation of
insulin action by glucose (36). Finally, Kellerer et al.
(24) have found that PKC-
has an inhibitory effect on
tyrosine kinase activity of the insulin receptor in human embryonic
kidney cells. Taken together, these data support the hypothesis that
FFAs induce hepatic insulin resistance through the activation of
PKC-
.
Finally, it should be noted that although hepatic insulin resistance was evident when the insulin levels were matched in the 7-h experiment, suppression of EGP was not significantly impaired by IH when the same insulin infusion rate was used as in control experiments. This suggests that the concomitant elevation in insulin levels (due to the decrease in hepatic insulin clearance) maintains suppression of EGP relatively intact, although the rise in insulin is not sufficient to prevent a decrease in peripheral glucose uptake.
In conclusion, we have shown that FFAs increase EGP in the basal state
and during hyperinsulinemic clamps. This effect was associated with at
least two mechanisms, an increase in FFA oxidation and/or PKC-
translocation. Furthermore, FFA induced a progressive impairment in
insulin suppression of EGP (i.e., hepatic insulin resistance) in
parallel with a progressive increase in PKC-
translocation. Thus
PKC-
translocation may be relevant to the pathogenesis of hepatic
insulin resistance in states associated with chronic FFA elevation such
as obesity and type 2 diabetes.
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ACKNOWLEDGEMENTS |
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We thank Drs. M. Prentki and S. Farfari for assaying the hepatic citrate content.
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FOOTNOTES |
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This work was funded by grants from the Canadian Diabetes Association (to A. Giacca) and the Canadian Institutes for Health Research (to I. G. Fantus).
T. K. T. Lam was supported by an Ontario Graduate Scholarship and a Univ. of Toronto Fellowship. E. Bogdanovic was supported by a Novo Nordisk-Banting and Best Diabetes Centre Studentship.
Part of this work was presented at the 61st Annual Meeting and Scientific Sessions of the American Diabetes Association, June 22-26, 2001, Philadelphia, PA.
Address for reprint requests and other correspondence: A. Giacca, Dept. of Physiology, Univ. of Toronto, Medical Science Bldg., 1 King's College Circle, Rm. 3363, Toronto, ON M5S 1A8, Canada (E-mail: adria.giacca{at}utoronto.ca).
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.
10.1152/ajpendo.00038.2002
Received 31 January 2002; accepted in final form 29 May 2002.
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