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Endocrinology Vol. 145, No. 11 5006-5012
Copyright © 2004 by The Endocrine Society

Intestinal Lipoprotein Production Is Stimulated by an Acute Elevation of Plasma Free Fatty Acids in the Fasting State: Studies in Insulin-Resistant and Insulin-Sensitized Syrian Golden Hamsters

Gary F. Lewis, Mark Naples, Kristine Uffelman, Nathalie Leung, Linda Szeto and Khosrow Adeli

Departments of Medicine and Physiology, Division of Endocrinology and Metabolism (G.F.L., K.U., N.L., L.S.), and Department of Laboratory Medicine and Pathobiology (M.N., K.A.), Hospital for Sick Children, University of Toronto, Toronto, Ontario, Canada M5G 1X8

Address all correspondence and requests for reprints to: Dr. Gary F. Lewis, Toronto General Hospital, 200 Elizabeth Street, EN11-229, Toronto, Ontario, Canada M5G 2C4. E-mail: gary.lewis{at}uhn.on.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
It is not known whether intestinal lipoprotein production is stimulated by an acute elevation of plasma free fatty acids (FFA). We examined the effect of an intralipid and heparin infusion on the intestinal lipoprotein production rate (PR) in insulin-sensitive [chow-fed (CHOW)], insulin-resistant [60% fructose (FRUC) or 60% fat-fed (FAT)], and insulin-sensitized [FRUC or FAT plus rosiglitazone (RSG)-treated] Syrian Golden hamsters. After 5 wk of treatment, overnight-fasted hamsters underwent in vivo Triton WR-1339 studies for measurement of apolipoprotein B48 (apoB48) PR in large (Svedberg unit, >400) and small (Svedberg unit, 100–400) lipoprotein fractions, with an antecedent 90-min infusion of 20% intralipid and heparin (IH) to raise plasma FFA levels approximately 5- to 8-fold vs. those in the saline control study. IH markedly increased apoB48 PR in CHOW by 3- to 5-fold, which was confirmed ex vivo in pulse-chase experiments in primary cultured hamster enterocytes. Oleate, but not glycerol, infusion was associated with a similar elevation of apoB48 PR as IH. In FRUC and FAT, basal (saline control) apoB48 PR was approximately 4-fold greater than that in CHOW; there was no additional stimulation with IH in vivo and only minimal additional stimulation ex vivo. RSG partially normalized basal apoB48 PR in FAT and FRUC, and PR was markedly stimulated with IH. We conclude that intestinal lipoprotein production is markedly stimulated by an acute elevation of plasma FFAs in insulin-sensitive hamsters, in which basal production is low, but minimally in insulin-resistant hamsters, in which basal production is already elevated. With RSG treatment, basal PR is partially normalized, and they become more susceptible to the acute FFA stimulatory effect.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THERE IS INCREASING evidence that apolipoprotein B48 (apoB48)-containing, intestinally derived lipoproteins are particularly atherogenic (1, 2, 3, 4, 5, 6). Although much attention has focused on documenting reduced clearance of intestinally derived lipoproteins from the circulation in certain dyslipidemic states and understanding its mechanism, there is far less appreciation for the factors that regulate intestinal lipoprotein production in health and disease. The intestine has traditionally been viewed as a passive organ with respect to lipoprotein production, with intestinal lipoprotein production rates (PRs) responding mainly to dietary fat ingestion and absorption. In contrast to the intestine, numerous hormonal and nutritional factors are known to influence hepatic lipoprotein production, not the least of which are fatty acid flux to the liver and hepatic insulin action (7, 8).

Hepatic very low density lipoprotein (VLDL) production is regulated to a large extent by plasma free fatty acid (FFA) flux to the liver (9, 10). Elevated FFA flux to the liver in insulin-resistant states and type 2 diabetes is believed to play an important role in the pathogenesis of the typical dyslipidemia of these conditions (7). FFAs have been shown to directly stimulate hepatocyte VLDL triglyceride (TG) synthesis and secretion in HepG2 cells (11, 12, 13, 14, 15, 16, 17) and cultured hepatocytes (18, 19) and in humans (20). It remains an open question to what extent the intestine also responds to plasma FFA elevation.

We have recently demonstrated in the insulin-resistant, fructose-fed hamster, that there is overproduction of apo B48-containing intestinal lipoproteins in both the fasted and the fed state (21), and others have reported an increase in intestinal lipoprotein production in the desert sand rat (22). Our findings and those of others (22, 23, 24, 25) suggest that intestinal cells, like hepatocytes, may actively regulate lipoprotein production in response to humoral factors in the postabsorptive state, thereby contributing to the dyslipidemia of conditions such as insulin resistance and type 2 diabetes.

In the present study we examined the effect of an acute elevation of plasma FFAs on intestinal lipoprotein production in insulin-sensitive (chow-fed), insulin-resistant (fructose- and fat-fed), and insulin-sensitized [fructose- and fat-fed, treated with rosiglitazone (RSG)] Syrian Golden hamsters. RSG is a member of the thiazolidinedione class of insulin-sensitizing agents and has specific peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) agonist activity (26). Hamster liver has been previously shown to exclusively secrete apo B100-containing lipoprotein particles, because no apoB mRNA-editing activity has been detected in the liver (27, 28). Our own previous work has confirmed these observations (29, 30). The tissue-specific expression of apoB100 (only in the liver) and apoB48 (only in the intestine) makes it possible to measure intestinally derived apoB48-containing lipoprotein particle production in vivo, because apoB48 is an essential structural component of intestinally derived chylomicrons and chylomicron remnants. Triton WR-1339 infusion blocks lipoprotein lipase-mediated clearance of TG-rich lipoproteins (TGL) by a detergent effect, i.e. by coating the TRL particle, thereby preventing LPL access to the particle. The slope of the line of TRL apo B48 concentration vs. time after Triton WR-1339 administration was used to determine the TRL apo B48 PR, as previously described (21, 31). Additional studies were carried out ex vivo in primary cultured enterocytes derived from similarly treated hamsters to confirm by a second experimental method that secretion of apoB48 lipoproteins increases after the acute elevation of plasma FFAs.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and study protocols
Male Syrian golden hamsters (Mesocricetus auratus) were purchased from Charles River (Québec, Canada). All animals were housed in pairs and were given free access to food and water. Hamsters were fed a normal chow diet for 7 d to allow acclimatization to the new environment and recovery from the stress of shipping. Animals were then placed on one of five treatment protocols: 1) normal chow for 5 wk [CHOW; n = 7 for saline infusion and n = 10 for intralipid plus heparin (IH) studies], 2) high fructose diet (hamster diet with 60% fructose, Dyets, Inc., Bethlehem, PA) for 5 wk (FRUC; n = 15 for saline infusion and n = 8 for IH studies), 3) high fructose diet for 5 wk with RSG (20 µmol/kg·d; GlaxoSmithKline, Philadelphia, PA) diluted in water and given once daily by gavage for the last 3 wk of the fructose feeding period (FRUC+RSG; n = 24 for saline infusion and n = 8 for IH studies), 4) high fat diet (hamster diet with 60% fat, Dyets, Inc.) for 5 wk (FAT; n = 10 for saline infusion and n = 17 for IH studies), or 5) high fat diet for 5 wk with RSG (20 µmol/kg·d) for the last 3 wk of the fat-feeding period (FAT+RSG; n = 10 for saline infusion and n = 9 for IH studies). Only the hamsters treated with RSG received daily gavage. The hamsters’ weights were monitored every week. At the end of the 5 wk, the animals either underwent the in vivo protocol described below or were killed for isolation of enterocytes for the ex vivo protocols. All animal protocols were approved by the animal ethics committee of the University Health Network.

In vivo determination of intestinal lipoprotein particle PRs during saline control or acute elevation of plasma FFAs
One day before these studies, femoral venous and arterial catheters were inserted as previously described (30). Hamsters in each treatment group were randomly assigned to undergo either a saline control study or an IH infusion study. The animals were fasted overnight for 16 h. On the morning of the study, a 90-min infusion of 0.9% normal saline or 20% intralipid (Baxter Corp., Toronto, Ontario, Canada) and heparin (20 U/ml; Organon Canada, Toronto, Ontario, Canada) was started at a rate of 25 µl/min to raise plasma FFA levels. After the 90-min infusion, and with the infusion of saline or IH continuing, a baseline blood sample was drawn, followed by an iv bolus of Triton WR-1339 (Sigma-Aldrich Corp., St. Louis, MO). After Triton WR-1339 administration, blood samples were drawn at 10 and 20 min (i.e. 100 and 110 min after the start of the IH infusion; the total blood volume withdrawn for the entire study was 1.2 ml). Svedberg unit (Sf) >400 (large TRL) and Sf 100–400 (small TRL) fractions were isolated by ultracentrifugation of plasma samples at 16 C in a TI 100.3 rotor with a Optima TLX ultracentrifuge (Beckman Coulter, Fullerton, CA). To isolate the large TRL, plasma samples were overlaid carefully with density 1.006 kg/liter solution and spun at 50,000 rpm for 5 min. The top 0.5 ml was defined as the Sf >400 fraction. The bottom fraction was then reconstituted in density 1.006 kg/liter solution and spun at 70,000 rpm for 23 min, and the top 0.5 ml was defined as the Sf 100–400 fraction. ApoB48 was quantified using 4–20% analytical SDS-PAGE (Invitrogen Life Technologies, Burlington, Ontario, Canada) as described previously (32). Large and small apoB48 and TG secretion rates were derived by multiplying the slope of the concentration increases in apoB48 and TG, respectively, over time by the intravascular distribution volume, estimated as 3.8 ml/100 g body weight (30).

Because of limitations of the total volume of blood that can be drawn from a hamster undergoing an in vivo Triton WR-1339 study, basal (fasting) and intralipid-infused biochemical parameters were determined in an additional set of hamsters treated in an identical fashion to that described above. Six hamsters in each of the five treatment groups (CHOW, FRUC, FRUC+RSG, FAT, and FAT+RSG) underwent an infusion of IH without Triton WR-1339 administration for determination of basal and postinfusion plasma FFA, glucose, insulin, and TG concentrations as well as large and small TRL and apoB48 concentrations.

To determine whether glycerol infusion alone stimulates apoB48 secretion, three additional CHOW animals were infused with a 2.2% glycerol in saline solution alone at 25 µl/min to mimic the exogenous glycerol delivery in the IH infusion experiments as described above. Triton WR-1339 was administered 90 min after a basal blood sample was drawn, and blood samples were again drawn at 100 and 110 min. The plasma samples were ultracentrifuged as described above, and the apoB48 PR was calculated.

An infusion of oleic acid [12% (vol/vol) emulsified with FFA-free BSA (Sigma-Aldrich Corp.), diluted 1:2 with saline, and infused at 10.8 µl/min] was administered to four additional CHOW hamsters to determine whether oleate itself stimulates apoB48 secretion. Blood was drawn after 90 min of oleate infusion, Triton WR-1339 was administered, and blood was collected at 100 and 110 min. The plasma samples were treated as described above.

Ex vivo protocols
For the ex vivo experiments, additional studies were conducted to determine whether, using an ex vivo experimental technique, an acute elevation of FFAs stimulates intestinal lipoprotein production. One day before these studies, femoral venous and arterial catheters were inserted as described previously (30). The animals were fasted overnight for 16 h. On the morning of the study, a 90-min infusion of either 0.9% normal saline or 20% intralipid and heparin (20 U/ml) was started at a rate of 25 µl/min. Hamsters were killed at the end of the 90-min infusion for isolation of viable adult villi from the small intestine, based on the method described by Perrault et al. (33) with some modification as reported recently (21).

Metabolic labeling of intact primary hamster enterocytes
Primary hamster enterocytes were used for pulse-chase experiments as previously described (21). Briefly, cells were preincubated in methionine-free DMEM (Sigma-Aldrich Corp.) for 30 min and pulsed with 50 µCi/ml [35S]methionine for 20 min. After the pulse, the cells were washed and chased with Williams E medium supplemented with 40 mM cold methionine. At various chase times, triplicate dishes were harvested, cells were lysed in solubilization buffer, and the lysates were used for immunoprecipitation as previously described (30). Immunoprecipitates were analyzed by SDS-PAGE and fluorography, essentially as previously described (34). ApoB48 bands, visualized by fluorography, were quantified by excision from the gel, digestion, and scintillation counting essentially as previously described (30).

Other laboratory methods
Glucose was determined on whole blood using a glucometer (Sure Step, One Touch LifeScan, Inc., Milpitas, CA). Plasma insulin concentrations were determined by RIA using a rat insulin kit from Linco Research, Inc. (St. Louis, MO). This assay had 100% cross-reactivity to hamster insulin, and the intra- and interassay coefficients of variation were 6.8% and 10.6%, respectively. Plasma FFAs were measured by a colorimetric method (kit supplied by Wako Industrials, Neuss, Germany). TG was measured using a colorimetric assay (Roche, Mannheim, Germany).

Statistical analysis
All values are reported as the mean ± SEM. For comparison of in vivo apoB48 secretion rates among CHOW, FRUC, FRUC+RSG, FAT, and FAT+RSG hamsters, ANOVA was performed, followed by post hoc analysis with Tukey’s test. For the ex vivo studies, apoB48 secretion rates in normal saline-infused hamsters were compared with apoB48 secretion rates in IH-infused hamsters using an unpaired t test. P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Plasma glucose, insulin, FFA, and TG concentrations at baseline and during the 90-min infusion of IH (Table 1Go)
Because of the limitation of the total volume of blood that can be drawn from a hamster undergoing an in vivo Triton WR-1339 study, the data shown in Table 1Go are from additional animals that underwent an IH infusion without the administration of Triton WR-1339 (n = 6 in each treatment group). Plasma glucose, insulin, FFA, and TG were assessed in the fasting state and during IH infusion (the infused values in Table 1Go represent the mean of the 90, 100, and 110 min samples after starting the IH infusion without the administration of Triton WR-1339). Although there were trends toward higher body weight, plasma TG, insulin, and glucose in FRUC and FAT and some correction of TG, insulin, and glucose with RSG treatment, as shown in Table 1Go, there were no significant differences among the treatment groups.


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  		TABLE 1. Mean body weights and fasting plasma TG, FFA, insulin, and glucose concentrations in the various treatment groups fasting and during IH infusion (Inf)

 
TG and apoB48 concentrations in the Sf >400 (large) TRL fraction and Sf 100–400 (small) fraction in the various treatment groups fasting and during IH infusion (Table 2Go)
Similar to the data shown in Table 1Go, those presented in Table 2Go are from additional animals that underwent an IH infusion without the administration of Triton WR-1339 (n = 6 in each treatment group, the same animals from which the data in Table 1Go were derived). As shown in Table 2Go, there was a marked increase in the TG and apoB48 concentrations of the large and small TRL fractions in all treatment groups after IH infusion. The only significant differences, however, were a higher fasting TG in the Sf 100–400 (small) TRL fraction of FAT compared with CHOW (P < 0.05) and apoB48 in the Sf 100–400 (small) TRL fraction of FRUC+RSG vs. CHOW (P < 0.05).


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  		TABLE 2. Mean TG and apoB48 concentrations in the Sf >400 (large) TRL fraction and Sf 100–400 (small) fraction in the various treatment groups fasting and during IH infusion

 
Triton WR-1339 studies for determination of in vivo intestinal lipoprotein production during acute elevation of plasma FFAs (Fig. 1Go)
ApoB48 secretion rates are illustrated for the Sf >400 fraction (large TRL) in Fig. 1AGo and for the Sf 100–400 fraction (small TRL) in Fig. 1BGo for CHOW, FRUC, FRUC+RSG, FAT, and FAT+RSG hamsters. The apoB48 PR markedly increased with IH infusion vs. saline in CHOW (Sf >400 fraction, P < 0.01; Sf 100–400 fraction, P = 0.03). In FRUC and FAT, apoB48 PR was already markedly elevated in the absence of IH (2- to 4-fold compared with CHOW), and there was no additional increase with IH. RSG partially normalized basal large and small intestinal particle PRs in FAT and FRUC. In RSG-treated hamsters, both large and small intestinal lipoprotein fractions were additionally stimulated with IH vs. saline (FRUC+RSG: Sf >400 fraction, P < 0.01; Sf 100–400 fraction, P < 0.01; FAT+RSG: Sf >400 fraction, P < 0.01; Sf 100–400 fraction, P = 0.01).



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  		FIG. 1. In vivo production of Sf >400 (large; A) and Sf 100–400 (small; B) TRL particle apoB48 in CHOW, FRUC, FAT, FRUC+RSG (FRUC+R), and FAT+RSG (FAT+R). Sf >400 (A) and Sf 100–400 (B) apoB48 secretion rates (in micrograms per minute) with ({square}) and without ({blacksquare}) an antecedent 90-min infusion of IH. For the Sf >400 fraction (A) saline infusion studies, apoB48 production was elevated in all groups compared with CHOW (+, P < 0.01). For the Sf 100–400 fraction saline infusion studies (B), apoB48 production was elevated in FRUC and FAT compared with CHOW (+, P < 0.01). In both lipoprotein fractions, the acute elevation of plasma FFAs markedly increased apoB48 PR compared with saline control studies in CHOW, FRUC+R, and FRUC+R hamsters (*, P < 0.01), but not in FRUC or FAT.

 
There was no difference between the glycerol only-infused group and the saline-infused chow-fed animals [Sf >400 fraction, 2.82 ± 1.07 µg/min for glycerol vs. 2.65 ± 0.59 µg/min for saline (P = not significant [NS]; Sf 100–400 fraction, 2.55 ± 0.95 µg/min for glycerol vs. 3.85 ± 0.72 µg/min for saline (P = NS)], indicating no effect of glycerol in stimulating Apo B48 secretion from the intestine (data not shown graphically).

The magnitude of oleate-stimulated apoB48 secretion was significantly greater than that seen with saline infusion and was similar to that seen with IH infusion. The secretion of apoB48 in the Sf >400 fraction in response to oleate infusion was 15.70 ± 5.72 vs. 2.65 ± 0.60 µg/min for saline-infused, chow-fed hamsters (P < 0.01) and 15.50 ± 1.85 µg/min for IH-infused, chow-fed hamsters (P = NS for oleate vs. IH studies). For the Sf 100–400 fraction, the apoB48 PR was 8.18 ± 2.34 µg/min for oleate-infused vs. 3.85 ± 0.72 µg/min for saline-infused animals (P < 0.05) and 9.99 ± 2.11 µg/min for IH-infused animals (P = NS for oleate vs. IH studies). These data together with the absence of stimulation of apoB48 secretion with pure glycerol infusion conclusively confirm that it is the FFA component of intralipid that directly stimulated intestinal lipoprotein production.

Studies of apoB48 secretion in primary cultured enterocytes (Fig. 2Go)
IH infusion before isolation of intestinal cells increased the production and secretion of apoB48 from primary cultured enterocytes compared with saline-infused control animals. In CHOW, there was a significant increase in intestinal apoB48 accumulation among lipid-infused animals after the pulse period (time zero) compared with controls (19,944 ± 1,895 cpm in IH-infused vs. 11,740 ± 1,602 cpm in saline-infused hamsters; P < 0.0003; Fig. 2AGo, left panel). The increased apoB48 accumulation during the pulse in intralipid-infused hamsters most likely reflects differences in cotranslational degradation during the pulse period and amounts to approximately 70% less degradation during the pulse in intralipid-infused intestinal enterocytes. There was also a noticeable increase in total apoB at both 1 and 2 h of chase in enterocytes derived from intralipid-infused hamsters, suggesting enhancement of intracellular apoB stability. ApoB48 levels in both cells and media were normalized for total cellular protein mass and the incorporation of [35S]methionine into total trichloroacetic acid-insoluble cellular and secreted proteins. These increases in apoB48 production were reflected by enhanced secretion of apoB48 from lipid-infused enterocytes (Fig. 2AGo, right panel). Lipid infusion resulted in significant increases in apoB48 secretion at 45 min (5,854 ± 1,247 cpm) and 90 min (8,085 ± 1,290 cpm) compared with infusion with saline (3,863 ± 108 cpm at 45 min and 3,744 ± 442 cpm at 90 min; P < 0.03 and P < 0.0008, respectively).



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  		FIG. 2. Ex vivo secretion of apoB48 by primary hamster enterocytes from CHOW, FAT, and FRUC hamsters with and without an antecedent 90-min infusion of IH: effect of RSG treatment (FRUC+RSG and FAT+RSG). Primary enterocytes were isolated from fasting CHOW, FAT, FRUC, FAT+RSG, and FRUC+RSG hamsters with an antecedent 90-min infusion of 0.9% normal saline ({diamondsuit} in A–E) or IH ({blacksquare} in A–E). Primary enterocytes were pulsed with [35S]methionine and chased for 0, 45, and 90 min. The medium samples and cell lysates collected at each chase time point were subjected to immunoprecipitation and then analyzed by SDS-PAGE and fluorography. A, Total apoB48 (left panel of A, reflecting apoB48 turnover) and apoB48 secretion (right panel of A) in CHOW enterocytes with or without lipid infusion. B and C, ApoB48 turnover and secretion in FAT hamster enterocytes with or without lipid infusion and/or RSG treatment. D and E, ApoB48 turnover and secretion in FRUC enterocytes with or without lipid infusion and/or RSG treatment. See Results for statistical significance between saline- and intralipid-infused studies for each of the five treatment groups.

 
We also examined the effect of intralipid infusion and RSG treatment on the production and secretion of apoB48 in both FAT and FRUC hamsters. No significant increase in intestinal apoB48 accumulation was observed at either 0 or 90 min chase after lipid infusion in FAT (Fig. 2BGo, left panel), with a small, but significant, increase observed at 45 min chase. Similarly, no change in apoB48 secretion was observed either at 0 or 90 min chase, with a minor increase in apoB48 at 45 min chase (Fig. 2BGo, right panel). Interestingly, however, lipid infusion resulted in significant increases in apoB48 production and secretion in fat-fed hamsters after treatment with RSG (P < 0.05 at both 45 and 90 min; Fig. 2CGo). ApoB48 production in fructose-fed hamsters was sensitive to lipid infusion with and without RSG treatment (Fig. 2Go, D and E). Lipid infusion did not affect apoB48 accumulation in FRUC at 45 min chase, but significantly increased apoB48 production at 90 min chase (P < 0.05) and increased apoB48 secretion at both 45 and 90 min chase (P < 0.05 for both; Fig. 2DGo). After RSG treatment (FRUC+RSG; Fig. 2EGo), lipid infusion caused a greater rise in apoB48 accumulation at 0 and 45 min chase compared with untreated lipid-infused hamsters (FRUC). ApoB48 secretion was also significantly increased at both 45 and 90 min chase in FRUC+RSG (P < 0.05). These data suggest that intestinal apoB48 production in FAT and FRUC enterocytes was partially sensitive to lipid infusion, and this sensitivity was additionally increased after RSG treatment.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have previously demonstrated that intestinal lipoprotein production is increased in fructose-fed (21) and high fat-fed (31), insulin-resistant hamsters. We have also previously shown that treatment with RSG, a member of the thiazolidinedione class of insulin sensitizers with specific PPAR{gamma} agonist activity, improved whole body and hepatic insulin sensitivity and ameliorated the overproduction of VLDL-apoB (35) and intestinally derived apoB48-containing lipoproteins (31). In the present study we examined the effect of an acute elevation of plasma FFAs on the intestinal lipoprotein PR in insulin-sensitive (CHOW), insulin-resistant (FRUC+FAT), and insulin-sensitized (FRUC+FAT+RSG) Syrian Golden hamsters. We found that intestinal lipoprotein production is markedly stimulated by an acute elevation of plasma FFAs in insulin-sensitive hamsters. Glycerol alone had no effect on apoB48 secretion, whereas oleate stimulated apoB48 secretion, indicating that the effect of IH was due to the elevation of FFAs released by lipolysis of the synthetic TG emulsion. Although RSG treatment partially normalized the basal intestinal lipoprotein overproduction in FRUC+FAT hamsters, the insulin-sensitized hamsters became more susceptible to the FFA-stimulating effect, with marked stimulation of intestinal lipoprotein production during the IH infusion. The secretion of intestinal apoB48-containing particles was examined ex vivo after an acute elevation of plasma FFAs, and similar results were obtained, with the exception of the FRUC group, which ex vivo, but not in vivo, demonstrated a small increase in apoB48 secretion with intralipid infusion. As with hepatic overproduction of apoB-containing lipoproteins (30), the ex vivo experiments in intestinal enterocytes showed that the increase in apoB48 production was associated with greater stability of intracellular apoB48. Differences ex vivo were not as substantial as those in vivo. These may relate to the effect of removing the primary enterocytes from their natural environment and culturing them without all the in vivo factors present in the whole animal (that may be necessary for full expression of the differences under various conditions).

The present study confirmed our previous observations in two animal models of nutritionally induced insulin resistance, fructose-fed (21) and fat-fed (31) hamsters, that apoB48 secretion is significantly elevated in the postabsorptive (basal) state. An interesting, and perhaps unexpected, observation in the present study was that the acute elevation of plasma FFAs had no additional stimulatory effect on apoB48 secretion in fructose- or fat-fed hamsters in vivo and only a minimal stimulatory effect ex vivo. This suggests either that intestinal lipoprotein production was already maximally or near maximally stimulated in the basal state in these insulin-resistant animals, with no capacity to increase further with additional stimulation by FFAs, or that the intestinal cells were less responsive to plasma FFAs. The former scenario is less likely, because the intestine has a marked capacity to esterify fatty acids and diglycerides absorbed from the intestinal lumen and to assemble and secrete TG-rich chylomicrons in response to the influx of ingested fat. We have previously shown that intestinal lipoprotein secretion in the postabsorptive state is closely linked with de novo lipogenesis, which is up-regulated in fructose-fed, insulin-resistant hamsters (21). Perhaps enterocytes in insulin-resistant animals in the postabsorptive state may be more dependent on de novo lipid synthesis and less dependent on plasma FFA availability. The precise biochemical mechanism accounting for this observed phenomenon will require extensive additional study.

In studies of insulin-sensitized hamsters, RSG reduced and partially normalized basal apoB48 secretion in fructose- and fat-fed hamsters, as we have previously shown (21, 31). The novel observation in the present study is that apoB48 production was markedly stimulated with intralipid and heparin in fructose- and fat-fed hamsters that had been treated with RSG, which suggests that RSG treatment sensitized the intestinal cells of these hamsters to the acute stimulatory effect of plasma FFAs. PPAR{gamma} agonists, such as rosiglitazone, are increasingly believed to exert their insulin-sensitizing effects by repartitioning fatty acids in the adipose tissue depot and away from insulin-responsive tissues (37). Although this is not always reflected in a reduction in fasting plasma FFA concentration (in the present study there was no difference in fasting plasma FFAs between treatment groups), FFA flux has been shown to be reduced with PPAR{gamma} treatment (38). We have previously shown that short-term (3-h) incubation of hamster enterocytes with RSG in vitro does not alter apo B48 secretion (unpublished observations), suggesting that RSG requires a systemic effect to exert its effect on intestinal lipoprotein production. We could not, however, rule out a possible direct effect of RSG on intestinal apoB48 particle secretion after longer periods of time because of the difficulties in maintaining intestinal cells in culture for prolonged periods.

Intestinal lipoprotein absorption is thought to be highly efficient, and the enterocyte has little control over the rate of entry of these fatty acids (39). The intestinal cell expresses two types of fatty acid-binding protein, the hepatic form and the intestinal form. The intestinal cell also has extremely active fatty acid activation and esterification enzymes located in the endoplastic reticulum (40), and the conversion of absorbed FAs to TG is very rapid, with 79% of the mucosal labeled fatty acid being esterified to TG within 30 sec (41, 42). During studies of intestinal secretion, Glickman et al. (43, 44) visualized apoB in isolated rat villus epithelial cells as early as 10 min after lipid exposure. These results are consistent with studies of lymph chylomicrons that demonstrate incorporation of apoB into lymph chylomicrons within 30 min of administration of the isotope (45). The intestine may also use plasma FFAs to synthesize and secrete lipoproteins (46), although plasma FFAs were shown to be preferentially oxidized and incorporated into phospholipids, in contrast to luminal fatty acids, which were preferentially esterified to triglyceride (47).

In conclusion, we have shown that intestinal lipoprotein production is markedly stimulated by an acute elevation of plasma FFAs in insulin-sensitive hamsters, in which basal production is low, but not in insulin-resistant hamsters, in which basal production is already elevated. With RSG treatment, the insulin-sensitized hamsters become susceptible to the FFA-stimulating effect. To the best of our knowledge, our studies are the first to compare the role of plasma FFAs in intestinal lipoprotein production in insulin-sensitive, insulin-resistant, and insulin-sensitized animals. Additional studies examining the link among plasma FFAs, insulin resistance, and intestinal lipoprotein overproduction should be conducted in humans to determine whether this process is relevant to human disease states.


   Footnotes
 
This work was supported by operating grants from Canadian Institutes for Health Research [MOP43839 (to G.F.L.) and MOP53093 (to K.A.)] and in part by an operating grant from GlaxoSmithKline Canada. N.L. was the recipient of a Research Fellowship from the Heart and Stroke Foundation of Canada/Canadian Institute of Health Research/Rx&D Research Program. G.F.L. holds a Canada Research Chair in Diabetes and is a Career Investigator with the Heart and Stroke Foundation of Canada.

Abbreviations: apoB48, Apolipoprotein B-48; CHOW, chow-fed hamsters; FAT, fat-fed hamsters; FFA, free fatty acid; FRUC, fructose-fed hamsters; IH, intralipid and heparin; NS, not significant; PPAR, peroxisome proliferator-activated receptor; PR, production rate; RSG, rosiglitazone; Sf, Svedberg unit; TG, triglyceride; TRL, triglyceride-rich lipoprotein; VLDL, very low density lipoprotein.

Received November 18, 2003.

Accepted for publication July 14, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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