ameliorated hepatic insulin resistance is associated with
TRANSCRIPT
Ameliorated hepatic insulin resistance is associated with normalization of microsomal triglyceride transfer protein expression and reduction in VLDL
assembly and secretion in the fructose-fed hamster.
André Carpentier* , Changiz Taghibiglou†, Nathalie Leung*, Linda Szeto* , Stephen Van
Iderstine†, Kristine Uffelman*, Robin Buckingham§, Khosrow Adeli†, and Gary F. Lewis* ¶
*Department of Medicine, Division of Endocrinology & Metabolism, University Health
Network, and †Department of Laboratory Medicine & Pathobiology, Hospital for Sick Children, University of Toronto, Toronto, Ontario, Canada§GlaxoSmithKline.
Running Title: Hepatic insulin sensitization and reduced VLDL secretion.
¶ To whom correspondence should be addressed:
Gary F. LewisDivision of Endocrinology & Metabolism,Toronto General Hospital200 Elizabeth St.Toronto, Ontario, CanadaM5G 2C4Phone: (416) 340-4270Fax: (416) 340-3314E-mail: [email protected]
Copyright 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
JBC Papers in Press. Published on June 4, 2002 as Manuscript M204568200 by guest on February 16, 2018
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SUMMARY
To determine whether reduction of insulin resistance could ameliorate fructose-induced
very-low density lipoprotein (VLDL) oversecretion and to explore the mechanism of this effect,
fructose-fed hamsters received placebo or rosiglitazone for 3 weeks. Rosiglitazone treatment led
to normalization of the insulin-mediated suppression of glucose production rate and to a ∼2-fold
increase in whole body insulin-mediated glucose disappearance rate (p < 0.001). Rosiglitazone
ameliorated the defect in hepatocyte insulin-stimulated tyrosine phosphorylation of the insulin
receptor, IRS-1, and IRS-2, and the reduced protein mass of IRS-1 and IRS-2 induced by
fructose feeding. Protein tyrosine phosphatase-1B levels were increased with fructose feeding
and were markedly reduced by rosiglitazone. Rosiglitazone treatment led to a ~50% reduction of
VLDL secretion rates (p < 0.05) in vivo and ex vivo. Despite this, fasting plasma triglycerides
were not significantly different with rosiglitazone treatment, although they tended to be reduced
in the latter (by ~30%, p = 0.16). VLDL clearance assessed directly in vivo was not significantly
different in the FR vs. F animals, although there was a trend towards a lower clearance with
rosiglitazone. Enhanced stability of nascent apolipoprotein B (apoB) in fructose-fed hepatocytes
was evident and rosiglitazone treatment resulted in a significant reduction in apoB stability. The
increase in intracellular mass of microsomal triglyceride transfer protein (MTP) seen with
fructose feeding was reduced by treatment with rosiglitazone. In conclusion, improvement of
hepatic insulin signaling with rosiglitazone, a PPAR γ agonist, is associated with reduced hepatic
VLDL assembly and secretion due to reduced intracellular apoB stability.
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INTRODUCTION
The typical dyslipidemia of insulin resistant states and Type 2 diabetes consists of
hypertriglyceridemia due to VLDL overproduction, low high-density lipoprotein-cholesterol
and small-dense low-density lipoprotein particles (1). Elevated plasma free fatty acid (FFA) flux
from peripheral and intra-abdominal adipose tissue depots, due to resistance to insulin’s anti-
lipolytic and esterification effect in adipose tissue, is felt to play an important role in driving
VLDL assembly and secretion in insulin resistant states (2-4). Nevertheless, previous studies in
humans have suggested that insulin also has an important direct effect on the liver in controlling
VLDL secretion (5-7).
Rat and mouse models of insulin resistance and type 2 diabetes have provided important
insights into the molecular mechanisms of insulin resistance. These animal models may not,
however, be ideal for the study of human lipoprotein disorders because, unlike humans, their
livers secrete apoB48 and apoB100-containing VLDL and they do not necessarily develop
VLDL oversecretion as the basis for their hypertriglyceridemia (8;9). Unlike livers from rat or
mouse, the liver of the golden Syrian hamster secretes only apoB100-containing VLDL and its
lipoprotein metabolism more closely resembles that of humans (10). We have shown that insulin
resistance in the fructose-fed golden Syrian hamster is associated with mild
hypertriglyceridemia, VLDL-apoB oversecretion, increased intracellular apoB-containing
lipoprotein particle stability and increased expression of microsomal triglyceride transfer protein
(MTP) (11). The present studies were conducted to explore the effect of improving insulin
sensitivity in this insulin resistant animal model by treatment with rosiglitazone, a peroxysome
proliferator-activated receptor gamma (PPAR-γ) agonist and insulin sensitizer, and to gain
further insight into the molecular mechanisms of VLDL oversecretion in insulin resistant states.
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EXPERIMENTAL PROCEDURES
Animals and study protocols
Male Syrian golden hamsters (Charles River, Quebec, Canada) were housed in pairs and
were given free access to food and water. After 7 days acclimatization animals were placed on a
fructose-enriched diet (hamster diet with 60% fructose, pelleted, Dyets Inc., Bethlehem, PA) for
5 weeks. After two weeks of feeding with the fructose-enriched diet, the animals were
randomized to receive either rosiglitazone (20 µmol/kg/day) (GlaxoSmithKline, PA, USA)
diluted in water vs. water only given once daily by gavage for the remaining three weeks of the
fructose feeding period. At the end of the 5 weeks, the fructose-fed (F) and fructose-fed +
rosiglitazone treated (FR) animals underwent either one of the three in vivo protocols described
below or isolation of hepatocytes for the ex vivo protocols. In addition, some animals remained
on regular chow for 5 weeks to serve as normal controls.
In vivo protocols
Euglycemic hyperinsulinemic clamp studies: Studies were performed as previously
described (11) with the following modifications. Catheters were kept patent overnight with 4%
heparin in normal saline (Hepalean, Organon Teknica, 1000 I.U./ml). At 8:00 am the morning
after insertion of femoral vein and arterial catheters a primed (10 µCi) constant (0.1 µCi/min)
infusion of HPLC-purified [3-3H]-glucose (New England Nuclear, Boston, MA) was started
(time –90 min) (12). [3-3H]-glucose was added to the 20% dextrose infusate to minimize the
decline in glucose specific activity during the clamp. After 75mins of equilibration at time 0
min, a primed (80 mU/kg) constant insulin infusion (8 mU/kg/min in 0.1% BSA in normal
saline) (Humulin R, Eli Lilly, Canada) was started and a D20% infusion was adjusted at 10-min
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intervals to maintain blood glucose at baseline level. Blood samples (0.25 ml) were taken from
the arterial line at times –15, 0, 90, 100, 110, and 120 min of the clamp for measurement of
blood glucose, [3-3H]-glucose SA, and plasma insulin levels. There was no significant decline
in hematocrit throughout the study. Endogenous glucose production (Ra) was calculated as the
endogenous rate of appearance measured with [3-3H]-glucose using a modified one-
compartment model (13). Insulin-mediated glucose disappearance (∆Rd) was the rate of
disappearance measured with [3-3H]-glucose during the clamp minus the mean baseline Rd
level. Data were smoothed with the optimal segments routine (14), using the optimal error
algorithm (15). Because euglycemia was not maintained in one hamster of the FR group, this animal
was not included in the analysis of these experiments.
In vivo VLDL secretion studies: One day prior to these studies, catheters were inserted
into the femoral vein and artery of F (n = 10) and FR animals (n = 9) of similar weight (134 ± 3 g
vs. 132 ± 2 g, respectively, p = 0.64) as previously described (11). VLDL-apoB and VLDL-
triglyceride (TG) secretion rates were measured in the fasting state (12 hours) after intravenous
injection of Triton WR-1339 (Sigma Chemical Co), as previously described (11). The total blood
volume of the samples drawn was less than 1.5 ml per animal during the experiment and there
was no significant decline in hematocrit.
In vivo VLDL clearance studies: Because the triton method does not allow direct
assessment of VLDL clearance, the following studies were performed after a 12 hour fast in 7 F
and 8 FR animals of similar weight (129 ± 6 g vs. 126 ± 4 g, respectively, p = 0.68). Catheters
were inserted the day prior to these studies into the femoral vein and artery. A bolus (20 µCi) of
[2-3H]-glycerol (New England Nuclear, Boston, MA) was injected intravenously and blood
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samples were collected at times 10, 15, 20, 25, 30, 35, 40, and 50 min after the injection to
measure VLDL-TG levels and to determine the rate of decline of VLDL-TG [3-3H]-glycerol
specific activity (SA). The fractional clearance rate of VLDL-TG (FCR in pool/min) was
assessed by the slope of the natural logarithm of VLDL-TG [2-3H]-glycerol SA over time
determined by linear regression over the linear portion of the down-slope, as previously
described (16). Ex vivo protocols
Liver Perfusion and Isolation of Primary Hamster Hepatocytes: After an overnight fast,
the liver of animals from the F and FR groups was perfused under anesthesia and hepatocytes
released from digested liver tissue were transferred into culture medium and seeded in collagen
coated plates as previously described (11).
Determination of ex vivo Tyrosine-Phosphorylation of Insulin Receptor, IRS-1 and
IRS-2 in Primary Hamster Hepatocytes.
In order to detect tyrosine phosphorylation of insulin receptor β subunit (IR), IRS-1, and
IRS-2, hepatocytes derived from fructose-fed and fructose-fed rosiglitazone-treated hamsters
were incubated for 5 h in serum and insulin free media. Cells were then stimulated with 100nM
insulin for 10 minutes at room temperature. Cells were lysed with a buffer containing
phosphatase inhibitor cocktail [150 mM NaCl, 10 mM tris (hydroxymethyl)aminomethane (pH
7.4), 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 1% NP-40, 2 mM PMSF, 10 µg/ml
aprotinin, 10 µg/ml leupeptin, 100 mM sodium fluoride, 10 mM sodium pyrophosphate, and 2
mM sodium orthovanadate and subjected to immunoprecipitation with specific polyclonal
antibodies (against insulin receptor β subunit or IRS-1) or a specific mouse monoclonal antibody
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against IRS-2 . Immunoprecipitates were used for immunoblotting with mAb αPY (1:1000
dilution) using ECL chemiluminescence system as described below.
Determination of ex vivo VLDL-apoB secretion in primary hepatocyte cultures:
Radiolabeled VLDL-apoB prepared from collected media by ultracentrifugation was subjected to
immunoprecipitation and SDS-PAGE and apoB band was quantified by liquid scintilation
counting as described (11).
Pulse chase of Primary Hamster Hepatocytes to assess nascent apoB particle stability:
We employed pulse-chase labeling experiments to assess the stability of apoB in hepatocytes
isolated from fructose-fed hamsters treated with rosiglitazone vs. placebo, as described
previously (17).
Chemiluminescent Immunoblotting: Cell samples were subjected to chemiluminescent
immunoblotting for the protein mass of the MTP 97 kDa subunit, as previously described (11). A
similar method was utilized to measure protein expression levels of IR, IRS-1, IRS-2, and PTP-
1B.
Other laboratory methods
Measurement of glucose, insulin, FFA, TG, apo B, [3-3H]-glucose SA and VLDL
isolation were performed as previously described (11) (7). VLDL-TG [2-3H]-glycerol SA (dpm/mg)
was determined as previously described (5).
Statistical analysis
All the values are reported as MEAN ± SEM unless otherwise stated. For the euglycemic
clamp studies, two-way ANOVA was used to compare the glucose, insulin, Ra, and ∆Rd curves
of the F, FR, and control chow fed groups at baseline and during the last 30 minutes of the clamp
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and difference between the three groups was assessed by post-hoc analysis using Scheffe test. A
two-tailed unpaired homoscedastic t-test was used to compare all the other quantitative
parameters between F and FR hamsters and between F and control chow fed were indicated. A p
value less than 0.05 was considered to be significant.
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RESULTS
Effect of rosiglitazone treatment on body weight, plasma insulin, FFA, TG and glucose (Table
1).
Due to constraints imposed by the small blood volume of the animals, not all variables
were measured on each animal undergoing the various experiments. Fasting plasma insulin was
significantly lower (p = 0.02) in the FR and control chow fed group than in the F group. Total
plasma TG levels tended to be lower (by ~30%, p = 0.16) following rosiglitazone treatment vs.
the fructose fed hamsters and were identical to TG levels in the control chow fed hamsters. All
other variables were not significantly different.
Treatment of fructose-fed hamsters with rosiglitazone ameliorates whole-body insulin
sensitivity and improves hepatocyte insulin signaling
1- Euglycemic hyperinsulinemic clamp studies:
Plasma glucose (Figure 1A) was higher in the F vs. FR animals at baseline (4.4 ± 0.3 vs.
3.2 ± 0.2 mmol/l, p = 0.03) and during the last 30 minutes of the clamp (4.0 ± 0.3 mmol/l vs. 3.0
± 0.1 mmol/l, p < 0.001), but was kept constant by design throughout the clamp. Hamsters fed a
normal chow diet had intermediate glucose levels at baseline (3.7 ± 0.2 mmol/l) and during the
last 30 minutes of the clamp (3.4 ± 0.1 mmol/l, p < 0.001 vs the F group). The insulin levels
(Figure 1B) were similar throughout the clamp in the F, FR, and the control chow fed group.
Glucose SA (not shown) remained constant in the last 30 minutes of the clamp in the three
groups. The endogenous glucose production rate (Ra) (Figure 1C) was significantly higher in the
F vs. FR animals at baseline (80.6 ± 12.2 vs. 54.0 ± 11.1 µmol/kg/min, p < 0.001) and
throughout the clamp (51.9 ± 14.3 vs. 10.7 ± 7.0 µmol/kg/min, p < 0.001). Treatment of the F
animals with rosiglitazone resulted normalization of Ra at baseline and during the clamp (p = NS
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vs control chow fed group) and also led to normalization of the level of suppression of Ra from
baseline (64.7 ± 15.6 vs. 19.1 ± 11.4 vs. 13.1 ± 8.4 % of baseline level during the clamp in the F,
FR, and control chow fed group respectively, p < 0.001 for the difference between F and the two
other groups). The glucose infusion rate (GINF) (not shown) was significantly lower in the F vs.
the FR group during the last 30 minutes of the clamp (64.7 ± 8.7 vs. 121.7 ± 25.1 µmol/kg/min, p
< 0.001). However, rosiglitazone treatment did not completely correct the GINF and remained
lower than control chow fed (GINF of control chow fed 176.2 ± 3.0 µmol/kg/min, p < 0.001 vs.
FR group). Consequently, insulin-mediated glucose disappearance rate (∆Rd) (Figure 1D)
during the clamp was also significantly lower in the F vs. FR animals (29.4 ± 8.4 vs. 75.3 ± 20.8
µmol/kg/min, p <0.001) but was not completely normalized by treatment with rosiglitazone (∆Rd
of control chow fed 119.6 ± 5.1 µmol/kg/min, p <0.001 vs. FR).
(place figure 1 here).
2- Insulin signaling in hamster primary hepatocyte cultures:
In hepatocytes isolated from F, insulin-stimulated insulin receptor β subunit tyrosine
phosphorylation was reduced to 34.1 ± 2.6% (n=3, p=0.033) of that in control hepatocytes
derived from chow-fed hamsters and this was restored to the control levels (98.3 ± 0.5%, n=3, p
= 0.01 vs. F) following rosiglitazone treatment, indicating complete restoration of insulin
receptor phosphorylation by the drug (Figure 2A). Insulin receptor appears as a doublet on the
gel. We have consistently observed this doublet in hamster hepatocytes. We do not believe that
the second band is a result of degradation since addition of protease inhibitors does not prevent
the detection of the doublet (data not shown). Insulin-stimulated IRS-1 phosphorylation vs.
basal was 184.3 ± 22.6% in control chow fed (n=4, p=0.002), 130.3 ± 5.3% in F (n=4, p=0.007),
and 188.9 ± 8.5% in FR (n=4, p=0.001) (Figure 2B), indicating improvement of IRS-1
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phosphorylation to the control levels in hepatocytes isolated from FR (p < 0.001 F vs. FR and p =
0.49 for control chow fed vs. FR). The effect of insulin on phosphorylation of IRS-2 was
similar to that of IRS-1, as shown in Figure 2C, indicating significant reduction (n=3, p=0.01 vs.
control chow fed) in insulin-stimulated IRS-2 phosphorylation with fructose feeding and a
marked improvement (n=3, p=0.004 vs. F) after treatment with rosiglitazone. As shown in Figure
3A, fructose feeding had no significant effect on IR protein mass (100 ± 14.1% in control chow
fed vs. 88.3 ± 29.6% in F, n=4, p=0.3). However, in FR hepatocytes, IR protein mass was
increased more than two-fold vs. cells derived from control chow fed and F animals (212.6 ±
47% of control chow fed, n=4, p=0.001 vs. F). Fructose feeding reduced protein mass of IRS-1
(Figure 3B) by 77% from 359.7 ± 23.9 scanning units/mg of total protein in hepatocytes from
control chow fed animals to 80 ± 11.5 in hepatocytes from F animals (n=3, p=0.0002 vs. control
chow fed). Rosiglitazone-treatment partially restored IRS-1 mass to 52.8 ± 11.1% of that in
control chow fed (n=3, p=0.003 vs. F). IRS-2 protein mass in hepatocytes isolated from F
hamsters was reduced to 57.8 ± 7.1% (p = 0.001) of the levels in control chow fed, whereas
rosiglitazone treatment increased it to 74.1 ±8 % of that of control hepatocytes (n= 4, p=0.002 vs.
F) (Figure 3C). These data suggest that the observed change in IR, IRS-1, and IRS-2
phosphorylation in hepatocytes isolated from FR may be partially due to change in protein
expression levels of these proteins.
Interestingly, PTP-1B protein mass increased to 169.9 ± 13.2% (n=3, p = 0.0002) of that
of controls with fructose feeding. FR had marked reduction of PTP-1B levels to 24.4 ± 12.9%
of that of control chow fed animals (n=3, p = 0.0004 vs. F) (Figure 3D).
(place figures 2 and 3 here).
Treatment of fructose-fed hamsters with rosiglitazone ameliorates VLDL-apoB and VLDL-TG
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oversecretion in vivo and ex vivo, without affecting VLDL clearance.
The slope of the increase in VLDL-apoB (Figure 4A) over time after injection of Triton
WR-1339 was significantly steeper in the F vs. FR group (2.42 ± 0.51 vs. 1.09 ± 0.27
µg/ml/min, p < 0.05) and vs. the control chow fed group (0.3 ± 0.1 µg/ml/min, p < 0.05).
Consequently, the VLDL-apoB secretion rate was higher in the F vs. FR group (12.4 ± 2.7 vs.
5.5 ± 1.4 µg/min, respectively, p < 0.05) and vs. the control chow fed group (1.3 ± 0.3 µg/min, p
< 0.05) (insert of Figure 4A). Similarly, VLDL-TG increase over time after injection of Triton
WR-1339 (Figure 4B) was significantly higher in F vs. FR hamsters (0.024 ± 0.004 vs. 0.011 ±
0.004 µmol/ml/min, respectively, p < 0.05) and vs. the control chow fed group (0.009 ± 0.002
µmol/ml/min, p < 0.05). The VLDL-TG secretion rate (insert of Figure 4B) was higher in the F
than in the FR group (0.12 ± 0.02 vs. 0.06 ± 0.02 µmol/min respectively, p < 0.05) and higher
than the control chow fed group (0.04 ± 0.01 µmol/min, p < 0.05). As depicted in Figure 4C,
rosiglitazone treatment significantly reduced ex vivo VLDL-apoB secretion to 38± 32% (mean ±
SD, n=4, p < 0.001) of that of fructose-fed hepatocytes, in keeping with the in vivo findings.
(place figure 4 here).
In vivo VLDL-TG FCR, as determined from the [2-3H]-glycerol bolus studies, was not
significantly different between the F vs. FR animals (0.034 ± 0.008 vs. 0.025 ± 0.004 min-1
respectively, p = 0.33), although clearance tended to be slightly delayed in the latter.
Treatment of fructose-fed hamsters with rosiglitazone leads to intracellular destabilization of
nascent VLDL particles and correction of enhanced expression of MTP
In pulse-chase labeling experiments, after one hour chase, there was a significant
reduction in the fraction of apoB secreted (Figure 5A) in hepatocytes from F vs. FR animals (88
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± 3% vs 49 ± 6% respectively, p=0.001). Decreased secretion was also accompanied with a
significant decrease in total apoB recovered (Figure 5B). There was also a significant reduction
in the fraction of labeled apoB secreted in the FR vs. F animals after 2 hour chase (53 ± 7% vs 97
± 1% in the FR vs F animals respectively, p=0.004) and similarly higher level of total apoB
recovered, suggesting that rosiglitazone treatment led to destabilization and increased
degradation of nascent apoB containing particles. The cellular protein mass of MTP in
hepatocytes from F was 153.3 ± 6.6% (n=4, p=0.0002) of that of controls (Figure 5C).
Rosiglitazone treatment led to normalization of cellular protein mass of MTP in fructose-fed
hamsters to 107.0 ± 9.4% of that of controls (n=4, p<0.005 vs. F).
(place figure 5 here).
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DISCUSSION
In the present study we have demonstrated that treatment of fructose-fed insulin-
resistant hamsters with rosiglitazone, a member of the thiazolidinedione class of insulin
sensitizers with specific PPAR-γ agonist activity, improved whole body and liver insulin
sensitivity in vivo, insulin signaling in the liver and reduced VLDL secretion in vivo and ex
vivo. Furthermore, rosiglitazone treatment was associated with a reversal of the increased expression
of MTP seen with fructose feeding and with de-stabilization of intracellular nascent apoB-
containing lipoproteins, indicating potential molecular mechanisms by which insulin
sensitization led to reduction of VLDL secretion in this insulin resistant animal model.
Treatment with rosiglitazone has been shown to improve glucose metabolism at least in
part by improving skeletal muscle insulin sensitivity in insulin-resistant humans (18) and animals (19).
This is consistent with the demonstration of increased whole-body glucose disposal rate with
treatment of fructose-fed hamsters in the present study. Rosiglitazone treatment has also
resulted in insulin sensitization of adipose tissue (20) and has often led to a reduction of plasma FFA
levels and flux (21;22). Although rosiglitazone treatment did not result in a significant reduction in
fasting plasma FFA in the present study, we cannot rule out that rosiglitazone treatment in this
model may have resulted in lower postprandial FFA levels and lower overall FFA flux to the
liver. If this were the case, reduced FFA flux to the liver could have accounted in part for the
reduced VLDL secretion with rosiglitazone treatment. More studies will be required to evaluate
this possibility.
Reduction of TG secretion with thiazolidinedione treatment has also been found in
sucrose-fed and obese Zucker rats by other investigators (22;23). Nevertheless, most published studies
in rats or mice did not show an inhibitory effect of thiazolidinediones on VLDL secretion,
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thereby concluding that the lowering of plasma TG resulted total or in part from increased
VLDL clearance (22;24;25). Unlike the fructose-fed hamster and insulin resistant humans, the rodent
models used in the latter studies display impaired plasma TG clearance as the major mechanism
of their hypertriglyceridemia when they become insulin resistant (8). This perhaps explains the
discrepancy between our results and those of the latter studies. Also, unlike the present study,
previous studies did not directly assess VLDL clearance. Whether rosiglitazone and other
thiazolidinediones can affect lipoprotein lipase expression and activity in animals and humans is
controversial, with some studies showing increased expression and activity (24;26) while others
showing either no effect (27) or even reduced expression and activity in adipose tissue (28).
A limitation of the tritiated glycerol method used in the present study to assess VLDL
clearance is that any change in de novo lipogenesis induced by treatment with rosiglitazone in
the present study could result in a change in the relative contribution of glycerol-derived
palmitate synthesis to VLDL-TG turnover, resulting in some error in the assessment of VLDL-
TG glycerol turnover. To our knowledge, no previous study has addressed whether treatment
with a thiazolidinedione results in alteration of fructose-induced elevation of in vivo hepatic de
novo lipogenesis. Although a putative effect of rosiglitazone on the induction of hepatic de
novo lipogenesis (29) may be expected to somewhat alter VLDL-TG glycerol turnover, de novo
lipogenesis contributes less than 20% of total VLDL-TG turnover in fructose-fed rodents(30).
Since only a fraction of hepatic de novo lipogenesis is derived from glycerol, it is unlikely that
any effect of rosiglitazone on de novo lipogenesis would significantly alter total VLDL-TG
glycerol turnover.
Treatment with thiazolidinediones has resulted in either no significant reduction or, at
best, a modest lowering of plasma triglycerides (TG) in clinical trials in humans with insulin
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resistance and Type 2 diabetes (31), despite their documented insulin sensitizing effects (21;32;33). This is
consistent with our observation that treatment with rosiglitazone resulted in a non-significant
reduction in fasting plasma TG levels in the fructose-fed hamster, an animal model of mild
hypertriglyceridemia associated with VLDL oversecretion. However, the mild
hypertriglyceridemia induced by fructose feeding was completely reversed by treatment with
rosiglitazone in the present study. A marked reduction of plasma TG levels after treatment with
thiazolidinediones has been more consistently shown in various mouse and rat models of insulin
resistance and type 2 diabetes, animal models that display a much more pronounced fasting
hypertriglyceridemia than the one usually found in insulin resistant humans (23;34;35) and in our hamster
model. In the present study, the reduction of VLDL secretion in the fructose-fed hamster
accounted for the reduction of plasma TG levels associated with rosiglitazone treatment, since
VLDL-TG clearance was not different with rosiglitazone treatment. In fact, the VLDL clearance
rate was slightly lower with rosiglitazone treatment vs. fructose alone, which could explain why
the 50% reduction of VLDL secretion observed both in vivo and ex vivo with rosiglitazone
treatment did not translate into a significant reduction in fasting plasma TG levels. To our
knowledge, the effect of treatment with rosiglitazone on VLDL production and clearance in
humans has not been reported.
In the present study, we documented definite improvement in the insulin-signaling
cascade in hepatocytes isolated from fructose-fed hamsters treated with rosiglitazone, as well as
a significant reduction of endogenous glucose production in vivo. Whether the improved hepatic
insulin sensitization in the present study resulted from a direct hepatic effect of rosiglitazone or
from an indirect effect, secondary to the action of rosiglitazone on extrahepatic tissues, is
unclear. We showed that primary hepatocytes from fructose-fed hamsters display a significant
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increase in PTP-1B expression, which was markedly reduced with rosiglitazone treatment.
PTP-1B has been shown to dephosphorylate the insulin receptor, and perhaps also IRS-1, and
plays a very important role in the regulation of insulin signaling (36). Increased PTP-1B expression
in skeletal muscle, adipose tissue, and liver has also been found in other animal models of
insulin resistance and diabetes (37-39) and in humans with obesity or diabetes (40;41). Knock-out mice for
this enzyme are very sensitive to insulin, are resistant to fat-induced insulin resistance, and
display an increased phosphorylation of liver and muscle insulin receptor after insulin injection (42;43).
We have recently shown that increased expression of PTP-1B precedes the reduction of
insulin-mediated tyrosine phosphorylation of IRS-1 and IRS-2 observed in primary hamster
hepatocytes with prolonged ex vivo exposure to high concentration of insulin (44). We have also
shown that incubation with vanadate, a general phosphatase inhibitor, leads to a dose-dependent
reduction in cellular and secreted apoB (44), a finding that has also been reported in primary rat
hepatocytes (45). To our knowledge, this is the first report of the effect of treatment with a
thiazolidinedione on PTP-1B expression. Clearly, this PTP-1B lowering effect of rosiglitazone
could be a very important potential mechanism for the liver’s insulin sensitizing effect of this
drug observed in our study. Further studies are needed to address whether this occurs as a direct
effect at the liver or secondary to changes induced in extra-hepatic tissues.
The reduction in MTP levels with rosiglitazone treatment may have been implicated in
the reduction of VLDL secretion in the present study. MTP plays an important role in VLDL
assembly and intracellular stabilization of apoB (46), although it may not be required for the late
lipidation of the particle (47). The promoter region of the MTP gene contains a negative insulin-
response element (48) and insulin, acting through its receptor, can lower MTP expression in HepG2
cells (49). Therefore, it is likely that the reduction in MTP levels induced by rosiglitazone treatment
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was a consequence of improvements in insulin signaling at the liver. However, the precise
molecular signaling pathway involved in insulin-mediated modulation of MTP expression is
currently unclear. Given the complexity of insulin’s regulation of VLDL secretion, it is unlikely
that modulation of MTP levels in the liver associated with insulin sensitization is the sole
explanation for the rosiglitazone-induced reduction of intracellular apoB-containing particle
stability and consequent VLDL secretion.
We have previously shown that fructose feeding results in increased apo B stability and
VLDL assembly in the Syrian Golden hamster (11). An important finding in the present study was
the reduction in nascent apo B stability with rosiglitazone treatment. We have recently shown
that approximately 40% of nascent apo B is degraded intracellularly in hamster hepatocytes (10).
Posttranslational apo B degradation is felt to be an important regulatory mechanism controlling
the rate of VLDL secretion (50). The factors regulating apo B degradation are complex but
hepatocyte lipid availability, insulin action and MTP activity are three important factors (50).
Rosiglitazone treatment could have reduced apo B stability in the fructose fed hamster by any
one of these mechanisms, ie by reducing FFA flux to the liver and hence reducing hepatocyte
triglycerides, by improving insulin action and hence increasing apo B degradation, or by
reducing MTP activity and hence reducing nascent VLDL particle assembly.
In conclusion, we have shown that whole-body and hepatic insulin sensitization with
rosiglitazone treatment is associated with a reduction in hepatic MTP expression, apoB stability,
and VLDL secretion in the fructose-fed insulin resistant hamster. Our findings suggest that
therapeutic measures that effectively ameliorate hepatic insulin sensitivity or that reduce MTP
overexpression in insulin resistant states could be part of the strategy to correct the VLDL
oversecretion associated with insulin resistance.
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ACKNOWLEDGEMENTS:
These studies were supported in part by operating grants from the Canadian Institutes of
Health Research, Heart and Stroke Foundation of Ontario, and GlaxoSmithKline. Dr. André
Carpentier was supported by a Heart and Stroke Foundation of Canada/Medical Research
Council cardiovascular research fellowship and is currently a New Investigator of the Canadian
Institutes of Health Research. Dr. Gary Lewis holds a Diabetes Research Chair from the
Canadian Institutes of Health Research and is a Scientist of the Heart and Stroke Foundation of
Canada.
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FIGURE LEGENDS:
Figure 1: Euglycemic hyperinsulinemic clamp: Blood glucose levels (A), plasma insulin levels
(B), endogenous glucose appearance rate (Ra) (C), and insulin-mediated glucose disappearance
rate (∆Rd) (D) during euglycemic hyperinsulinemic clamp studies from time 0 to 120 min in
fructose-fed hamsters treated with rosiglitazone (open circles, n = 5) vs. placebo (closed circles,
n = 6) vs. hamsters fed a chow diet (control, open squares, n = 5). Bars represent mean ± SEM.
Figure 2: Insulin-mediated phosphorylation of the insulin receptor (IR), IRS-1 and IRS-2:
Each panel depicts a representative immunoblot along with combined densitometric quantitation
of multiple experiments performed in duplicate or triplicate. Net intensity of the bands was
normalized for the total protein content of the samples. Insulin-mediated phosphorylation of the
insulin receptor (n =3) (A), IRS-1 (n = 3) (B), and IRS-2 (n = 4) (C) in hepatocytes from control
hamsters fed regular chow, and from fructose-fed hamsters treated with rosiglitazone vs. placebo
(n = 3 to 4 per experiment). All data are shown as mean ± SD.
Figure 3: Protein Mass of IR, IRS-1 and IRS-2: Representative immunoblots along with
combined densitometric quantitation of 3 to 4 experiments performed in duplicate or triplicate
for IR (A), IRS-1 (B) IRS-2 (C), and PTP-1B (D) respectively. Net intensity of the bands was
normalized for the total protein content of the samples and is either expressed as scanning
unit/mg total protein (panel B) or percent of control cells (panels A, C and D). Solid, open, and
gray bars represent IR, IRS-1, and IRS-2 protein mass in control chow fed, fructose-fed, and
fructose-fed + rosiglitazone-treated hepatocytes, respectively. All data are shown as mean ±
SD.
Figure 4: VLDL-apoB and VLDL-TG secretion rates: In vivo VLDL-apoB (A) and VLDL-TG (B) levels over
time after intravenous injection of Triton WR-1339 (600mg/kg) in fructose-fed hamsters treated with rosiglitazone
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(light gray circles, n = 10) vs. placebo (closed circles, n = 9) and control chow fed (dark gray squares, n = 5).
Inserts in (A) and (B) are showing VLDL-apoB and VLDL-TG secretion rate, respectively, in the rosiglitazone
(light gray bars), placebo-treated group (closed bars) and in the control chow fed (dark gray bars). Ex vivo VLDL-
apoB secretion rate (C) in hepatocytes derived from fructose-fed hamsters treated with rosiglitazone (open bars, n =
4) vs. placebo (closed bars, n = 3). Data are shown as mean ± SD.
Figure 5: Pulse-chase labeling experiments to assess the stability of apoB in hepatocytes from
fructose-fed hamsters treated with rosiglitazone: Distribution of immunoprecipitable apoB in
media (secreted apoB) (A). Immunoprecipitable apoB remaining in cells+media (total apoB) (B).
The fructose-fed + rosiglitazone treated (closed circles) vs. fructose-fed + placebo treated group
(open circles) expressed as a percentage of radiolabeled apoB at time 0. *Significantly different
from fructose-fed hepatocytes (secreted apoB; p=0.001 at 1h, p=0.004 at 2 h). **Significantly
different from fructose-fed hepatocytes (total apoB; p=0.001 at 1h, p=0.0095 at 2 h) (n=3).
Microsomal triglyceride transfer protein (MTP) expression (C). Data are shown for hepatocytes
from control hamsters fed regular chow, and from fructose-fed hamsters treated with
rosiglitazone vs. placebo as indicated (n = 4 per group, p<0.005 for the difference between
fructose-fed + rosiglitazone vs. fructose-fed + placebo animals). The MTP bands were
quantitated by densitometric scanning and the mass of the 97 kDa MTP subunit detected was
expressed as a percentage of the MTP mass detected in control cells. Please note that the blot
shows the result of one representative experiment whereas the graph displays the mean ± SD of 4
independent experiments. They are not therefore exactly the same. Data are mean ± SD.
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FOOTNOTES: ABBREVIATIONS USED
ApoB: apolipoprotein B
F: Fructose-fed + placebo-treated hamsters
FFA: free fatty acids
FR: Fructose-fed + rosiglitazone-treated hamsters
MTP: microsomal triglyceride transfer protein
IRS-1: insulin receptor substrate-1
IRS-2: insulin receptor substrate-2
PMSF: phenylmethylsulfonylfluoride
PPAR γ : peroxysome proliferator-activated receptor gamma
PTP-1B: protein-tyrosine phosphatase-1B
Ra: endogenous glucose appearance rate
∆Rd: insulin-mediated glucose disappearance rate
SA: specific activity
SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis
TG: triglyceride
VLDL: very low density lipoprotein
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TABLE 1: Characteristics of Fructose fed + placebo (F) vs. Fructose fed +
Rosiglitazone treated (FR) hamsters, mean (SEM).
F n FR n Controls n
∆ Weight (g) 34 (1) 51 36 (2) 52 30 (3) 19
FFA (mmol/l) 0.625 (0.061) 13 0.550 (0.097) 13 0.689 (0.139) 9
Total TG (mmol/l) 2.06 (0.36) 11 1.40 (0.24) 9 1.40 (0.24) 9
Insulin (pmol/l) 331 (38) 26 221 (25)* 24 140 (25)* 14
Glucose (mmol/l) 4.3 (0.3) 23 3.9 (0.3) 22 3.5 (0.1) 14
FFA: plasma free fatty acid concentration. * p < 0.05 vs. F group.
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A) B)
C) D)Time (min)
-20 0 20 40 60 80 100 120
Pla
sma
glu
cose
(m
mo
l/l)
0
1
2
3
4
5
6
7
Fructose + placebo (n = 6)Fructose + rosiglitazone (n = 5)Control (n = 5)
Time (min)
-20 0 20 40 60 80 100 120
Ra
( µm
ol/
kg/m
in)
0
20
40
60
80
100
120
Time (min)
-20 0 20 40 60 80 100 120
Pla
sma
insu
lin
(p
mo
l/l)
0
500
1000
1500
2000
2500
3000
Time (min)
-20 0 20 40 60 80 100 120
∆Rd
(µm
ol/
kg/m
in)
0
50
100
150
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0
20
40
60
80
100
120
140
Control F FRSti
mu
late
d i
ns
uli
n r
ec
ep
tor
tyro
sin
ep
ho
sp
ho
ryla
tio
n (
% o
f c
on
tro
l) P < 0.05 P = 0.001
Insulin
Control F FR
+ + +
A)
B)
+ +Insulin
Control
F
FR
Ph
os
ph
ory
late
d I
RS
-1
(Sc
an
nin
g U
nit
s /
mg
To
tal
Pro
tein
)
0
10000
20000
30000
40000
50000
60000
70000
Control F FR
- Insulin + Insulin
C)
+ +Insulin
Control
F
FR
0
100
200
300
400
500
Control F FR
Sti
mu
lato
ry IR
S-2
T
yro
sin
e P
ho
sph
ory
lati
on
(% o
f co
ntr
ol)
P = 0.01 P < 0.005
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0
50
100
150
200
250
300
Control F FR
Insu
lin R
ecep
tor
Pro
tein
Mas
s
(per
cen
t o
f co
ntr
ol)
P= 0.001P= 0.3
A
050100150200250300350400450
Control F FR
P < 0.001P < 0.02
IRS
-1 P
rote
in M
ass
(Sca
nn
ing
Un
its/
mg
To
tal P
rote
in)
B
0
50
100
150
200
Control F FR
PT
P-1
B P
rote
in M
ass
(% o
f co
ntr
ol)
P < 0.001 P < 0.001
D
020
40
60
80
100
120
Control F FR
IRS
-2 P
rote
in M
ass
(P
erce
nt
of
con
tro
l) P= 0.001
P= 0.002
C
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A)
B)Time (min)
0 20 40 60 80 100
∆V
LD
L a
po
B (µg
/ml)
0
50
100
150
200
250
300
Fructose + placebo (n = 9)Fructose + rosiglitazone (n = 10)Control (n = 5)
VL
DL
ap
oB
se
cret
ion
(µg
/min
)
0
5
10
15
Time (min)
0 20 40 60 80 100
∆V
LD
L T
G (
mm
ol/l
)
0.0
0.5
1.0
1.5
2.0
2.5
3.0
VL
DL
TG
se
cre
tio
n (µm
ol/
min
)
0.00
0.05
0.10
0.15
0
20
40
60
80
100
120
F FR
Rad
iola
bele
d V
LD
L-a
poB
sec
rete
d(%
of
cont
rol)
P < 0.001
C)
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Total ApoB
Imm
un
opre
cip
itat
ed R
adio
lab
eled
Ap
oB (
% o
f ze
ro t
ime)
BSecreted ApoB
Imm
un
opre
cip
itat
ed R
adio
lab
eled
A
poB
(%
of
zero
tim
e)
A
Control F FR
0
20
40
60
80
100
120
140
160
Control F FR
MT
P P
rote
in M
ass
(rel
ativ
e to
con
trol
)
P < 0.005
C
0%
50%
100%
150%
200%
0 1 2Chase time (h)
Fructose
Fruc+Rosi
0%
50%
100%
150%
0 1 2
Chase time (h)
*
**
*
**
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Iderstine, Kristine Uffelman, Robin Buckingham, Khosrow Adeli and Gary F. LewisAndré Carpentier, Changiz ;Taghibiglou, Nathalie Leung, Linda Szeto, Stephen Van
secretion in the fructose-fed hamstertriglyceride transfer protein expression and reduction in VLDL assembly and
Ameliorated hepatic insulin resistance is associated with normalization of microsomal
published online June 4, 2002J. Biol. Chem.
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