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J. Biol. Chem., Vol. 279, Issue 18, 19362-19374, April 30, 2004

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Regulation of Hepatic Apolipoprotein B-lipoprotein Assembly and Secretion by the Availability of Fatty Acids

I. DIFFERENTIAL RESPONSE TO THE DELIVERY OF FATTY ACIDS VIA ALBUMIN OR REMNANT-LIKE EMULSION PARTICLES^*

Yuan-Li Zhang, Antonio Hernandez-Ono, Carol Ko, Koichi Yasunaga, Li-Shin Huang, and Henry N. Ginsberg¶

From the Department of Medicine, Columbia University College of Physicians and Surgeons, New York, New York 10032

Received for publication, January 9, 2004

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The in vivo effects of increased delivery of fatty acids (FA) to the liver are poorly defined. Therefore, we compared the effects of infusing either 6 mM oleic acid (OA) bound to albumin, 0.5-20% Intralipid, or saline for 3 or 6 h into male C57BL/6J mice. Infusions were followed by studies of triglyceride (TG) and apoB secretion. Although plasma FA levels increased similarly after either 20% Intralipid or 6 mM OA, TG secretion increased only after infusion of 4-20% Intralipid; TG secretion was unchanged by 6 mM OA. By contrast, 6-h infusions of either 6 mM OA or 4-20% Intralipid increased apoB secretion. 6 mM OA and 20% Intralipid each increased secretion of apoB from primary hepatocytes ex vivo. Importantly, 0.5-2% Intralipid, which delivered more FA to the liver than 6 mM OA, did not stimulate apoB secretion. Hepatic apoB mRNA levels were unaffected by either 6 mM OA or 20% Intralipid, but microsomal triglyceride transfer protein mRNA was significantly lower after 6-h infusions with 6 mM OA versus either saline or 20% Intralipid. Lower microsomal triglyceride transfer protein mRNA levels were associated with reduced hepatic TG mass after 6-h infusions of 6 mM OA. We conclude that 1) increased FA delivery to the liver in vivo increases secretion of apoB-lipoproteins via post-transcriptional mechanisms, 2) OA-induced apoB-lipoprotein secretion occurred at least in part via mechanisms other than by providing substrate for TG synthesis, and 3) the route of delivery of FA is important for its effects on apoB secretion.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Regulation of the assembly and secretion of apolipoprotein B (apoB)-lipoproteins and, in particular, very low density lipoprotein (VLDL),¹ by the liver has been studied intensively for many years in cultured hepatocytes (1-4). Many laboratories have also conducted in vivo studies of apoB-lipoprotein secretion in humans and animal models (5-7). However, despite the presence of a large body of work, several questions remain incompletely answered.

First and foremost is the question of whether increased flux of plasma fatty acids (FA) to the liver can stimulate VLDL secretion. Studies in cultured liver cell lines, primary hepatocytes, and perfused livers have provided conflicting results relative to the role of FA delivery in VLDL secretion. Thus, several (8-13), but not all (14-16) studies in cultured liver cell lines support the proposal that apoB secretion is increased by increased FA uptake. Studies in primary hepatocytes from fed rats (16-18) or hamsters (19, 20) have failed to show that exogenous FA stimulate secretion of apoB. However, we were able recently to demonstrate oleic acid (OA)-induced apoB secretion in primary hepatocytes from fasted mice (21). Results from perfused rat livers have also been mixed; OA had no effect on apoB secretion in chow-fed rats (22) but increased apoB secretion in fasted (22) or high carbohydrate diet-fed (23) rats. In vivo studies in humans have also been inconclusive. Lewis et al. (24, 25) demonstrate that, when they increased the plasma levels of FA by infusing Intralipid and heparin intravenously into normal humans, they were able to stimulate VLDL production. By contrast, Malmstrom et al. (26) were unable to increase VLDL apoB secretion when they raised plasma levels of FA by giving subjects only heparin by intravenous infusion for 8 h. Defining the role of FA in stimulating apoB secretion is critical because of the potential link between insulin resistance at the level of adipose tissues, increased release of adipocyte-derived FA into the circulation, and increased TG accumulation in non-adipose organs, including the liver (27, 28). Increased FA flux, as observed in insulin-resistant states, has been linked to increased secretion of VLDL particles in humans (5, 29-33).

Among other questions remaining unanswered are the following. 1) Is metabolic channeling of FA into VLDL TGs dependent on the origin of the FA? This is an issue that is just beginning to be addressed (34, 35). Thus, will FA delivered by albumin have the same fate as FA delivered in lipoproteins or FA generated by de novo lipogenesis? More specifically, are FA from each of these sources equally available for assembly into lipoproteins? 2) Do FA only act as a substrate for core and surface lipids of apoB-lipoproteins, or can they act as signaling molecules, stimulating specific steps in the transport of apoB through the secretory pathway? Our recent work in cultured liver cell lines supports a signaling role for FA (36). 3) What are the mechanisms whereby the liver maintains lipid homeostasis in the face of increasing or decreasing demands related to the secretion of TG-rich apoB-lipoproteins? Will increased FA availability always lead to increased assembly and secretion of apoB-lipoproteins, and will decreased FA availability provide signals leading to decreased secretion of apoB-lipoproteins?

These unanswered questions inspired us to develop an experimental approach that would allow us to study in more detail the in vivo effects of increased hepatic FA availability on both the secretion of apoB-lipoproteins from the liver and the response of the liver to changes in apoB secretion. In our first studies we addressed the following two hypotheses; first, that in vivo FA flux to the liver is an important determinant of the assembly and secretion of apoB-lipoproteins, and second, that the in vivo effects of FA on secretion of apoB, like those in cultured liver cells, are post-transcriptional. By infusing either 6 mM OA bound to albumin or varying concentrations of the lipid emulsion, Intralipid, via chronic jugular vein catheterization, we found that increasing plasma FA levels by either 6 mM OA or 4-20% Intralipid infusion increased the secretion of newly synthesized apoB through post-transcriptional mechanisms. Unexpectedly, 4-20% Intralipid increased the concomitant secretion of TGs, whereas the effect of 6 mM OA was only on apoB secretion. Importantly, 0.5-2% Intralipid, which delivered more TG-FA to the liver than 6 mM OA, did not stimulate apoB secretion. Although Intralipid is composed of TGs carrying several different FA as well as phospholipid and glycerol, the disparate results we observed suggest that the effect of albumin-bound OA on apoB secretion was related to its route of delivery. Furthermore, Intralipid at concentrations less than 4% did not stimulate either apoB or TG secretion despite the fact that those concentrations would have delivered more FA to the liver than would the infusion of 6 mM OA. These results have provided us with several new insights regarding the assembly and secretion of apoB-lipoproteins.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Chemicals—The animals studies were male C57BL/6J mice, age 12-14 weeks, purchased from The Jackson Laboratory, Bar Harbor, ME. All mice were maintained on a 12-h light/dark cycle (light cycle was 7 a.m. to 7 p.m.) and on a regular rodent chow diet.

Surgical Procedures—Mice were anesthetized with 3.3 µl/g body weight of ketamine (15 mg/ml) and xylazine (3 mg/ml). The incision, location of the jugular vein, insertion of the cannula, and closing of the wound were carried out with a dissection microscope after the operative procedure developed for rats (37). In brief, silicone rubber tubing (catalog number 11-189-15A, 0.51-mm inner diameter x 0.94-mm outer diameter, Fisher) filled with saline was inserted into the jugular vein, and the outer portion of the tubing was tunneled subcutaneously, exiting at the nape of the neck. The length of tubing inserted into the jugular vein was 5 mm (38). Once the tubing was in place, it was filled with saline to maintain patency and was closed at the end with a metal stopper. The mice were allowed to recover for 2-3 days before the experiment was performed. The tubing was flushed several times during that period.

Lipid, Glucose, and Insulin Determinations—Total plasma TG concentrations were measured using commercial kits (INFINITY^TM Triglyceride Reagent, Sigma). Plasma-free FA levels were measured by a colorimetric method using a commercial kit (number 994-75409) from Wako Chemicals (Richmond, VA). Plasma insulin concentrations were measured by enzyme-linked immunosorbent assay using a commercial kit (Mercodia, ultrasensitive rat insulin ELISA, American Laboratory Products Co., Windham, NH). Glucose levels were measured using an enzymatic kit (number 315-100; Sigma-Aldrich).

Infusion Study—The mice were randomly assigned to receive intravenous infusions of 6 mM OA (Sigma) bound to fatty acid-free albumin (the ratio of OA to bovine serum albumin was 5:1), 0.5, 1, 2, 4, 6, 10, or 20% Intralipid (Abbott), or saline. Albumin-bound OA was prepared as previously described (9). Intralipid was composed of (by weight) 20% soybean oil, 1.2% phospholipid, 2.25% glycerol, 76.55% water. The fatty acid composition of the soybean oil was: C16:0, 10.4%; C18:0, 4.0%; C18:1, 23.5%; C18:2, 53.5%; C18:3, 8.3%.

On the morning of the experiment, food was removed, and the silicone tubing was flushed with saline and connected through polyethylene tubing to a Harvard Compact Infusion Pump (Harvard Apparatus, Holliston MA). The infusion line ran inside a tether and through a swivel, which was suspended from the top of the cage. This procedure protected the infusion tubing and allowed the mice complete freedom of movement. The infusions were carried out at the rate of 2.5 µl/min for either 3 or 6 h. Blood samples were obtained from the retro-orbital plexus before and at various time points during the infusions for measurement of plasma TG, free fatty acids, glucose, and insulin.

Determination of in Vivo TG and ApoB Secretion Rates (Turnover Study)—After each 3- or 6-h infusion, in vivo secretion rates of TG and apoB were determined as described previously (39). Mice were injected intravenously with 500 mg/kg Triton WR1339 (25301-02-4, Sigma-Aldrich) in 0.9% NaCl. Plasma VLDL clearance is completely inhibited in mice under these conditions (40), and the accumulation of VLDL lipids and apolipoproteins in plasma after injection of Triton can be used to estimate rates of secretion of VLDL into the plasma compartment. Blood samples were collected at the end of infusion (0 min, preinjection) and at 30, 60, 90, and 120 min after injection of Triton for determination of TG levels, with the rate of rise of TG in plasma over time indicative of the rate at which TG was secreted from the liver (41).

ApoB secretion rates were measured by injecting each mouse with 200 µCi of [³⁵S]methionine (1175 Ci/mmol, PerkinElmer Life Sciences) together with Triton. Blood samples were taken at 60 and 120 min after injection, and the accumulation of [³⁵S]methionine-labeled apoB was used to determine the rates of VLDL apoB secretion. In these experiments, 5 µl of whole plasma samples were subjected to 4% SDS-PAGE, and the gel was dried and exposed to x-ray films to visualize labeled apoB. The apoB bands on the film were scanned for densitometry measurements. In prior studies we demonstrated that the direct application of plasma to the gel provided results that were equivalent to results obtained by first isolating VLDL by ultracentrifugation before gel electrophoresis (39).

ApoB Secretion from Primary Hepatocytes—In three separate experiments hepatocytes were prepared after 6-h infusions (without Triton WR 1339 or [³⁵S]methionine) according to the method of Berry and Friend (42). Livers were perfused through the portal vein with oxygenated Hanks' solution (Hanks' balanced saline solution, Invitrogen, catalog number 14175-095) buffered with 10 mM Hepes (Invitrogen, catalog number 15630106) and 5 mM Ca²⁺ at 37 °C, pH 7.4, containing 30 mg of collagenase (Type I, 245 units/mg, Worthington). Protease inhibitor mixture (No. 18h580, Roche Diagnostics) was added as well. Hepatocytes were plated into collagen-coated, 6-well plates at a cell density of 2.5 x 10⁶ hepatocytes/9.6 cm² in methionine- and cysteine-free Dulbecco's modified Eagle's medium (Grand Island Biological Co, Santa Clara, CA) that was supplemented with 10% fetal bovine serum. Cells were incubated for 2 h at 37 °C with 5% CO_2. Prior studies in our laboratory showed that apoB secretion was linear for 2 h.

After a 2-h incubation cells were washed 3 times with phosphate-buffered saline, and 1 ml of fresh Dulbecco's modified Eagle's medium of the same composition supplemented with 1.5% fatty acid free bovine serum albumin was added into each well. [³⁵S]Methionine (1175 Ci/mmol, PerkinElmer Life Sciences) was added (100 µCi/ml) to medium, and cells were stably labeled for 2 h at 37 °C with 5% CO₂. After labeling, medium was transferred to a tube containing a protease inhibitor mixture (final concentrations: 1 mM benzamidine, 5 mM EDTA, 0.86 mM phenylmethylsulfonyl fluoride, 100 kallikrein-inactivating units/ml of aprotinin, and 10 mM Hepes, pH 8.0), and cells were lysed by adding ice-cold lysis buffer (150 mM NaCl, 5 mM EDTA, 50 mM Tris, pH 7.4, 0.0625 M sucrose, 0.5% Triton X-100, and 0.5% sodium deoxycholate) containing 50 µg/ml leupeptin and 50 µg/ml pepstatin A (Peninsula Laboratories, Inc, Belmont, CA) in addition to the protease inhibitor mixture. ApoB in medium and cell lysate was immunoprecipitated according to our previously described method (9), and precipitates were analyzed by 4% SDS-PAGE. Incorporation of ³⁵S into apoB48 and apoB100 was assessed by autoradiography (X-AR, Eastman Kodak Co.). The final results were normalized using the protein mass and trichloroacetic acid-precipitated counts of cell lysate and relevant medium used for apoB immunoprecipitation. Protein masses of samples were determined with a BCA kit (Pierce).

Determination of Liver TG Levels—At the end of either 3- or 6-h infusions (without Triton WR 1339), livers from the mice were collected for the measurement of hepatic TG content. Total liver lipids were extracted by a modification of the Folch extraction (43). Briefly, snapfrozen liver tissues (150 mg) were homogenized and extracted twice with a chloroform:methanol (2:1 v/v) solution. The organic layer was dried under nitrogen gas and resolubilized in chloroform. An aliquot of this Folch extraction was resuspended in an aqueous solution containing 2% Triton X-100 (44) for the determination of TG mass. [¹⁴C]Triolein (Amersham Biosciences) was added to each sample before lipid extraction to estimate the percent of recovery, and final TG concentrations were adjusted accordingly. Total liver protein was extracted using T-PER^TM tissue protein extraction reagent (78510, Pierce). Protease inhibitor mixture (1873580, Roche Diagnostics) was added into the liver protein extraction to prevent protein degradation. Liver TG levels were expressed as µg of TG per mg of liver protein.

Determination of the Amount of Fatty Acids Delivered to the Liver by Intralipid and OA Infusion—To investigate the relative delivery of FA to liver during infusions of Intralipid or OA, 0.5% Intralipid labeled with [¹⁴C]triolein or 0.15 mM albumin-bound OA labeled with [³H] OA were injected rapidly through the jugular vein after 4.5-h of infusion with unlabeled 20% Intralipid or 6 mM OA, respectively. The choice of 0.5% Intralipid and 0.15 mM OA was based on our goal of introducing quantities of each source of FA into the circulation by bolus injection that would approximate the quantities delivered during 1 min of the infusion of 20% Intralipid and 0.5% OA. The choice of 4.5 h as the time for injection of the tracers was based on our preliminary studies showing steady state levels of plasma TG and FA at that time. The 0.5% Intralipid labeled with [¹⁴C]triolein and 0.15 mM OA labeled with [³H]OA were prepared as described previously (45). In brief, to label Intralipid, 0.15 µCi of [¹⁴C]triolein (Amersham Biosciences) was added to a small glass vial and dried with N₂ gas. One ml of 0.5% Intralipid was added to the vial and mixed thoroughly, the emulsion was sonicated three times on ice for 20 s each at the power setting of 40 W using a Branson Sonifier Cell Disruptor (model W185) (Branson Scientific, Inc., Plainview, NY) to incorporate the [¹⁴C]triolein into the core of the emulsion particle, and the resulting emulsion was stored at 4 °C before use in the experiment. To label OA, 60 µCi of [³H]oleic acid (PerkinElmer Life Sciences) was added to a small vial and dried under N₂ gas. One ml of OA bound to albumin was added to the vial and mixed thoroughly, and the resulting solution was rocked overnight at 4 °C. Before injection the solution was diluted with saline to reach the final concentration of 0.15 mM oleic acid. At the end of the 4.5-h infusions of either 20% Intralipid or 6 mM OA, the appropriate tracer was injected through jugular tubing. Five minutes after injection mice were sacrificed and cleared of blood by flushing with 20 ml of phosphate-buffered saline through the left ventricle. The organs (heart, lung, liver, spleen, and kidney) and tissues (muscle and fat) were collected and homogenized in 5 ml of phosphate-buffered saline using a Polytron Tissue Disruptor (Kinematica AG, Littau-Lucerne, Switzerland). Muscle was collected from the gastrocnemius and femoris sites, and adipose tissue was collected from the epididymis. One-ml aliquots of the homogenates were added to 3.5 ml of scintillation fluids, and radioactivity was counted to determine tissue uptake. Tissue uptake was expressed as the percentage of total recovered radioactivity, including from blood, at the end of experiment. Fat and muscle mass were calculated as 15 and 42% of body weight, respectively.

RNA Probe Preparation and RNase Protection Assays—Total cellular RNA was isolated from livers using TRIzol reagent following the protocol provided by the company (Invitrogen). The RNA probe for mouse apoB (46) was generated by amplification of the target gene from liver RNA (male C57 BL/6J mice) by reverse transcription-PCR. The probe for MTP was described previously (39). PCR primers used and the size of amplified products for each probe are shown in Table I. PCR products were cloned into a PCRII vector using a TA cloning kit obtained from Invitrogen. DNA sequences of these clones were verified by DNA sequencing using a PerkinElmer ABI 377 automatic DNA sequencer.

Statistical Analysis—The mean and S.D. are presented. Statistically significant differences (i.e. p < 0.05; two-tailed) in mean values among more than two groups were determined by one-way analysis of variance. Differences in the mean values between two groups were assessed by Student's t test.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
We conducted preliminary studies to determine the optimal concentration of Intralipid as well as the time course of changes in plasma FA and TG in response to infusions of OA and Intralipid. Our goal was to increase plasma FA levels to high physiological levels, about three times above base line, for several hours. We infused Intralipid at concentrations ranging from 0.5 to 20% for 6 h through a cannulated jugular vein. The results, depicted in Fig. 1A, demonstrate that concentrations of Intralipid between 0.5 and 10% did not increase plasma FA levels significantly; infusion of 20% Intralipid, which delivered 172 mg of TG-FA into the circulation over 6 h, raised plasma FA levels more than 2-fold compared with saline (0.80 ± 0.16 versus 0.30 ± 0.12 mmol/liter; p < 0.01) (n = 3 per group for 0.5-10% Intralipid; n = 8 for 20% Intralipid; n = 13 for saline). 6 mM OA was the highest concentration we could use without adversely affecting the mice, and that concentrations satisfied our need in terms of raising plasma FA levels. Indeed, a 6-h infusion of 6 mM OA raised plasma FA levels to 1.08 ± 0.32 mmol/liter (n = 9) (Fig. 1), and this was significantly greater than the FA level achieved by saline. Plasma FA concentrations tended to be higher after 6 mM OA versus 20% Intralipid infusions, but the levels were not significantly different from each other (p = 0.07). The finding that infusion of 0.5-10% Intralipid over 6 h, which delivered between 4.3 and 86 mg of TGFA into circulation (compared with the 1.5 mg of FA delivered by infusion of 6 mM OA), did not increase plasma FA levels suggested that 1) much of the FA generated by peripheral lipolysis of Intralipid TG was internalized in the adipose and muscle beds, where lipoprotein lipase was active, and/or 2) most of the Intralipid TG was taken up by tissues together with the whole Intralipid particle.

View larger version (29K): [in this window] [in a new window]   FIG. 1. Effects of intravenous infusions of 6 mM OA and Intralipid on plasma FA and TG levels. Plasma FA levels were measured at base line and the end of 6-h infusions of saline, 6 mM OA, and 0.5-20% Intralipid (A). At base line, the mean plasma FA levels were comparable in all the groups and were 0.2-0.3 mmol/liter. After 6-h infusions, FA levels were significantly increased in 6 mM OA and 20% Intralipid groups to 1.08 and 0.8 mmol/liter, respectively (p < 0.05 both versus saline). Lower concentrations of Intralipid did not increase plasma FA levels significantly versus saline. The time course for changes in plasma FA during the 6-h infusions of OA and 20% Intralipid was examined next. Plasma FA levels were measured at base line and 1, 2, 3, and 6 h after starting the infusions (B). Plasma FA levels increased during the first 1 h of infusion of either OA or Intralipid; FA levels remained stable during the OA infusion but drifted down slightly during the last few hours of the Intralipid infusion. There was also an increase in FA levels during the first 1 h of the saline infusion, but those levels returned to base line between 3 and 6 h. Only Intralipid infusion caused a significant increase in plasma TG levels over time (C). TG levels rose during the first hour of 20% Intralipid infusion and remained constant thereafter. TG levels during infusion of 6 mM OA were not different from saline. IL, Intralipid. The asterisk denotes significant differences (p < 0.05) compared with saline at the end of 6-h infusion.

There were no differences in the base-line (pre-infusion) concentrations of TG, FA, glucose, or insulin among the 6 mM OA-, 20% Intralipid-, and saline infusion-treated mice (Table II). In particular, base-line plasma FA levels were comparable among the three groups (OA, 0.24 ± 0.15 mmol/liter; Intralipid, 0.20 ± 0.08 mmol/liter; saline, 0.23 ± 0.14 mmol/liter). Although human studies have indicated that acute elevations of plasma FA can stimulate insulin secretion (47) and cause insulin resistance (48), there were no significant changes in either plasma insulin or plasma glucose levels at the end of the infusions in the mice receiving either OA or Intralipid compared with mice receiving saline (Fig. 2).

View larger version (12K): [in this window] [in a new window]   FIG. 2. Neither OA nor Intralipid infusion caused significant changes in plasma insulin and glucose levels compared with saline. Plasma insulin (A) and glucose (B) concentrations were measured in three groups (saline, n = 11; 6 mM OA, n = 9; 20% Intralipid, n = 6) at base line and at the end of infusions. The values represent the average concentrations of plasma insulin (ng/ml) and glucose (mg/dl) in each group. Open bars, at base line. Closed bars, at the end of infusion. IL, Intralipid.

View larger version (23K): [in this window] [in a new window]   FIG. 3. Infusion of 20% Intralipid, but not 6 mM OA, increased TG secretion in both 3- and 6-h infusions. The mice were infused with saline, 6 mM OA, and 20% Intralipid for 3 (A) or 6 h (B). At the end of the infusions, Triton WR1339 was injected intravenously. Blood samples were collected at base line, the end of infusion (just before injection of Triton, 0 min), and 30, 60, 120 min after Triton injection to measure plasma TG concentrations. In the graphs on the left of A and B, the mean plasma TG concentrations (mg/dl) were plotted against time. At the end of the infusions (0 min; before injection of Triton), the plasma TG levels were significantly higher in the Intralipid group than in the OA and saline groups. Plasma TG levels increased steadily in all three groups over the 120 min after the injection of Triton, but only 20% Intralipid significantly increased TG secretion compared with saline infusion. The same data expressed as the absolute increase in plasma TG levels between 30 and 120 min after Triton are shown in bar graphs on the right. TG base line, TG at base line before infusions. TG 0, TG at the end of the infusions just before Triton injection. The asterisk denotes significant differences (p < 0.05) between 20% Intralipid and both OA and saline infusions after 3- and after 6-h infusions.

View larger version (19K): [in this window] [in a new window]   FIG. 4. There was no significant increase in secretion of 35S-labeled apoB100 and apoB48 after 3 h of infusion of either 6 mM OA or 20% Intralipid. The effect of 3-h infusions of 6 mM OA or 20% Intralipid on secretion of newly synthesized apoB100 (A) and apoB48 (B) into plasma is depicted relative to saline (n = 6/group). Plasma 35S-labeled apoB levels at 60 and 120 min after Triton and [35S]methionine injection were determined by 4% SDS-PAGE, autoradiography, and densitometry scan. The relative levels of newly secreted plasma 35S-labeled apoB are presented as a percentage of newly secreted plasma apoB in saline-infused mice.

View larger version (18K): [in this window] [in a new window]   FIG. 5. Infusion of either 6 mM OA or 20% Intralipid for 6 h significantly increased secretion of 35S-labeled apoB100 and apoB48. The effect of 6-h infusions of 6 mM OA (n = 12) or 20% Intralipid (n = 8) on secretion of newly synthesized apoB100 (A) and apoB48 (B) into plasma are depicted relative to saline (n = 17). Plasma 35S-labeled apoB levels at 60 and 120 min after Triton and [35S]methionine injection were determined as described previously. The relative mean values of plasma 35S-labeled apoB are shown in the percentage of saline-infused mice on the y axes. The asterisk denotes significant differences (p < 0.05) relative to saline.

View larger version (19K): [in this window] [in a new window]   FIG. 6. Infusion of either 6 mM OA or 20% Intralipid increased secretion of newly synthesized apoB100 and apoB48 from primary hepatocytes. Immediately after 6-h infusions, primary hepatocytes were isolated, incubated, and radiolabeled for 2 h with [35S]methionine. Radiolabeled apoB100 and apoB48 levels were isolated in the media (A) and in the cells (B) by immunoprecipitation, and their levels were determined by 4% SDS-PAGE and autoradiography. Three separate, independent experiments were conducted, and the results were pooled for statistical analysis (nine wells in each group). The relative levels of 35S-labeled apoB are shown as the percent of saline control on the y axes. One asterisk denotes significant differences (p < 0.05) in comparison to saline infusion; two asterisks denote significant differences (p < 0.05) compared with Intralipid infusion.

View larger version (18K): [in this window] [in a new window]   FIG. 7. Infusion of neither OA nor Intralipid affected apoB gene expression in the livers of C57BL/6J mice. At the end of the Triton studies, total cellular RNA samples were isolated from the livers of the mice infused with saline (n = 5), OA (n = 4), and Intralipid (n = 4). These were used to measure mouse apoB mRNA levels by RNase protection assays. In A, each bar represents the mean level and S.D. from 4-5 mice from each group. The radioactivity of protected fragments derived from the test probe was normalized to the internal reference mouse cyclophilin mRNA levels. The mRNA levels derived from the mice infused with saline (control) were averaged and expressed as 100%. The mRNA levels of the mice infused with either OA or Intralipid were expressed relative to control values. B shows a representative PAGE/urea gel of mouse apoB mRNA and reference mouse cyclophilin mRNA.

View larger version (34K): [in this window] [in a new window]   FIG. 8. After 6-h infusions, livers from mice infused with 6 mM OA had less TG than livers from mice infused with either 20% Intralipid or saline. At the end of 3- or 6-h infusions (without Triton WR 1339), mice infused with 6 mM OA, 20% Intralipid, and saline were sacrificed, and the livers were collected for the measurement of liver TG content. The liver TG content from 3-h infusions (n = 3/group) is shown in A, and that from 6-h infusions (n = 13, 11, 11 in saline, OA, and Intralipid groups, respectively) is shown in B. The mean values of liver TG content are expressed as µg of TG/mg of liver total protein. The asterisk denotes significant differences (p < 0.05) in both comparisons to Intralipid and saline infusion.

View larger version (23K): [in this window] [in a new window]   FIG. 9. Differential hepatic uptake of OA and Intralipid. Tissue uptake of radiolabeled OA and Intralipid-triolein was determined after 4.5-h infusions of 6 mM OA and 20% Intralipid. After 4.5 h the infusions were stopped, and radiolabeled OA and Intralipid carrying radiolabeled triolein were immediately injected in quantities equal to those infused over 1 min. The animals were sacrificed 5 min after injection of the radiolabeled tracers. Tissue uptake was expressed as percent of total recovered radioactivity. Fat and muscle mass were calculated as 15 and 42% of body weight, respectively. Values are the means ± S.D. (n = 3 for each group).

View larger version (27K): [in this window] [in a new window]   FIG. 10. Dose response of Intralipid infusion. To investigate the effects of more similar quantities of FA delivered by OA or Intralipid on TG and apoB secretion, Intralipid infusions with different concentrations were carried out. After 6-h infusions, TG and 35S-labeled apoB turnover studies were carried out as described. A shows the mean value of TG secretion between 30 and 120 min after Trion injection. B shows the relative amount of 35S-labeled apoB secreted during the 2 h after injection of [35S]methionine in combination with Triton. A, *, p < 0.05 for TG secretion after Intralipid compared with the saline group. B: *, p < 0.05 for apoB100 secretion after Intralipid compared with saline; #, p < 0.05 of apoB48 secretion after Intralipid compared with saline group. IL, Intralipid.

View larger version (39K): [in this window] [in a new window]   FIG. 11. Six-hour infusions of 6 mM OA induced a significant decrease in MTP mRNA levels in livers of C57BL/6J mice. As described in Fig. 7, total liver cellular RNA from mice infused with saline, 6 mM OA, and 20% Intralipid for 3 h (n = 4/group) or 6 h (n = 9/group) were subjected to RNase protection assays to determine the levels of mouse MTP mRNA. A shows MTP mRNA levels from 3-h infusions, and B shows that from 6-h infusions in three groups. The bar graphs represent the mean levels and S.D. derived from each group of mice as calculated from gels such as the one shown on the left. Mouse cyclophilin mRNA level was used as the internal reference. The asterisk denotes significant differences (p < 0.05) in both comparisons to saline and Intralipid infusion.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In the studies reported here we directly infused either OA or Intralipid to increase delivery of plasma FA to the liver. Our results demonstrate for the first time in vivo a direct link between increased plasma FA delivery and the stimulation of apoB secretion into plasma. Thus, 6-h intravenous infusions of either 6 mM OA or 20% Intralipid in C57BL/6J mice, which raised plasma FA concentrations to similar, high physiologic levels, increased the secretion of newly synthesized apoB100 and apoB48 about 3 times compared with infusions of saline. The effects of both OA and Intralipid on in vivo secretion rates were confirmed by ex vivo experiments with primary hepatocytes that were isolated and studied immediately at the end of the 6-h infusions. The increased in vivo secretion of newly synthesized apoB was not associated with changes in apoB mRNA levels. This latter result indicates that the effects of OA and Intralipid on assembly and secretion of VLDL were post-transcriptional, supporting data generated from cultured liver cells (2-4, 49). In cultured liver cells, post-transcriptional regulation of apoB secretion actually begins co-translationally when a "decision" is made that determines whether apoB is ubiquitinylated and degraded by the proteasome (55, 56) or completes translocation and assembles into a lipoprotein particle (2). Additional, post-translational degradation pathways are also important in regulating apoB-lipoprotein secretion (2, 57). The exact site(s) where the delivery of plasma FA effects apoB secretion remains to be determined. Beyond demonstrating the link between delivery of plasma FA to the liver and apoB secretion, our results offered several additional insights into the roles that hepatic FA and TG availability play in regulating the assembly and secretion of apoB-lipoproteins. First, among these new insights was that delivery of only a very small quantity of albumin-bound FA (less than 1 mg, based on the quantity of OA delivered into the circulation over 6 h by infusion of 6 mm OA and the data in Fig. 9) could stimulate increased secretion of apoB without affecting secretion of TG. This finding indicates that in vivo apoB secretion can be uncoupled from the delivery of large quantities of substrate for hepatic TG synthesis. The uncoupling between increased apoB secretion, and the availability of increased TG for co-secretion was associated with a loss of TG from the liver; hepatic TG mass was reduced by 30% in livers from mice infused with OA for 6 h compared with saline-infused mice. The importance of uncoupled apoB secretion as the cause of reduced hepatic TG content is supported by our finding that, after 3 h of 6 mM OA infusion, at a time when apoB secretion had not yet been increased, the TG content of the liver had not yet been reduced. We were surprised that, in the absence of increased TG secretion (as measured by the Triton method), hepatic TG content was reduced between 3 and 6 h of OA infusion (when increased apoB secretion occurred). We must assume that the Triton methodology was not sensitive enough to pick up small changes in TG secretion that accompanied increased secretion of apoB-lipoprotein. Alternatively, increased oxidation of hepatic FA or decreased hepatic lipogenesis during the 6-h infusions of OA together with increased secretion of apoB-lipoproteins could have reduced hepatic TG mass. Preliminary studies of the expression of genes involved in FA oxidation (CPT1, AOX) or lipogenesis (ACC, FAS), however, showed no effects of infusion of either 6 mM OA or 20% Intralipid.

We previously demonstrated in cultured HepG2 and Mc RH7777 cells that OA could stimulate the assembly and secretion of VLDL even though TG synthesis and MTP activity were inhibited (36), leading us to conclude that OA acted as a signaling molecule to allow completion of the translocation of lipid-poor apoB and its subsequent fusion with a pre-existing lipid droplet in the secretory compartment (1, 36, 58, 59). Our present data support a signaling role for OA in vivo but differ from the tissue culture results in that OA was unable to stimulate the concomitant secretion of TG. We initially thought that the different experimental protocols used in the present study versus our previous work (36) might account for the differing outcomes. In the previous cell culture experiments, we exposed those cells to OA for a relatively short period of time. Thus, we only looked at a small window of time during which pre-existing lipid droplets within the secretory pathway would have been available for fusion with the increased numbers of newly synthesized apoB molecules moving through the secretory pathway. By contrast, in the present in vivo experiments we exposed the livers to OA for several hours, raising the possibility that the secretion of VLDL was increased during the early part of the OA infusion (mimicking our prior in vitro results), but that later, during the majority of the infusion period, the lack of an adequate supply of FA to stimulate TG synthesis led to an isolated increase in apoB secretion. However, the finding that 3-h infusions of OA did not stimulate apoB secretion does not support this hypothesis, although we cannot rule out the possibility that secretion of TG increased transiently at some point between 3 and 6 h of OA infusion.

We gained several additional insights regarding regulation of the assembly and secretion of TG-rich apoB-lipoproteins from our studies with a wide range of Intralipid. First, those studies indicated that the way FA is delivered to the liver is an important determinant of its effect on apoB secretion. Thus, when 0.5, 1, or 2% Intralipid were infused for 6 h, 1, 2, or 4 mg of TG-FA was delivered to the liver. ApoB secretion, however, was not stimulated. This result strongly suggests that the delivery of TG-FA by Intralipid was not qualitatively equal to the delivery of less than 1 mg of albumin-bound OA, at least in terms of the ability to stimulate apoB secretion. Only when 4-20% Intralipid were infused for 6 h (which would have delivered between 8.5 and 43 mg of TG-FA to the liver) was apoB secretion-stimulated. We believe that the most likely explanation for these observations is that lipolysis of Intralipid TG-FA by lipoprotein or hepatic lipase was only a minor source of FA delivery to the liver. If 0.75 mg of albumin-bound OA was required to stimulate apoB secretion, then it appears that this critical quantity of albumin-bound FA was not available for hepatic uptake until at least 4% Intralipid, which delivered a total of about 8.5 mg of TG-FA to the liver, was infused. This would mean that only about 10% of Intralipid TG-FA delivered to the liver during that infusion arrived as albumin-bound FA after lipolysis in the circulation by either lipoprotein or hepatic lipase. This is compatible with the finding that plasma FA levels did not rise until 20% Intralipid was infused. Although it is likely that lipoprotein lipase-associated lipolysis occurred during infusion of Intralipid (as indicated by the increase in plasma FA when 20% Intralipid was used), it appears that such lipolysis was associated with efficient targeting of the liberated FA to the local tissue bed. It is also likely that hepatic lipase generated FA in the liver circulatory bed, leaving us to suggest that either this is a minor pathway or that hepatic lipase-associated lipolysis of Intralipid generated FA that were taken up by a pathway that was not linked to apoB secretion. Future studies in which lipoprotein and hepatic lipase are inhibited are planned to address these issues.

If very little Intralipid TG-FA reached the liver as albumin-bound FA, then the bulk of the TG-FA that was taken up by the liver must have been internalized as particle-associated lipid, probably after the emulsion particles had acquired apoE to become "remnant-like." Why, then, was this source of FA unable to stimulate apoB secretion until the amounts taken up reached were about 10x the amount of albumin-bound OA that was taken up? A possible explanation is that lysosomal hydrolysis of internalized particulate TG generates FA that are unable to act as signaling molecules in the apoB secretory pathway. It has been demonstrated both in cell culture (60) and in vivo (61) that remnant lipoproteins can increase apoB secretion from the liver. In those studies, however, the delivery of TG-FA probably exceeded a critical level, similar to the conditions in which 4-20% Intralipid was infused. Indeed, our finding that 6-h infusions of 4-20% Intralipid stimulated both apoB and TG secretion suggest that Intralipid TG-FA were preferentially targeted for incorporation as a core lipid into apoB-lipoproteins.

A final, related insight from our studies relates to what has been referred to as the two-step pathway of VLDL assembly (1, 2, 58, 59). Our data support this scheme and suggest that the first step of initial apoB transport and limited lipidation may be regulated differently than the second bulk lipidation step. Specifically our studies suggest that although only a small increase in the flux of albumin-bound OA to the liver was required to stimulate apoB secretion, much larger quantities of FA had to be delivered before TG secretion was stimulated. Although the stimulation of apoB secretion by Intralipid only occurred at concentrations of the emulsion that concomitantly stimulated TG secretion, we do not believe that this contradicts two separate and unique mechanisms for stimulating the assembly and secretion of apoB-lipoproteins. Indeed, a 3-h infusion of 20% Intralipid also stimulated TG secretion but did not affect apoB secretion. Integrating all of these data, we believe that Intralipid was inefficient at delivering FA via a pathway that mimicked albumin-bound OA in terms of effects on apoB secretion; higher concentrations of Intralipid achieved delivery of adequate FA via that pathway (or into the same pool to which albumin-bound OA was targeted) and thereby stimulated apoB secretion. The additional TG-FA delivered to the liver by higher concentrations of Intralipid did not further stimulate apoB secretion but were available for incorporation into the additional apoB-lipoproteins that were being secreted. Whether the Intralipid-derived FA that was resecreted with apoB entered the liver as albumin-bound FA after lipolysis of Intralipid TG by lipoprotein or hepatic lipase, as TG that was hydrolyzed in hepatic lysosomes, or via both pathways remains to be determined.

We appreciate that Intralipid differs in several ways from albumin-bound OA and that a potential role of the other FA, such as palmitic acid and linoleic acid, must be considered. We cannot rule out that palmitic and/or linoleic acid are targeted for incorporation as TG into newly assembled VLDL. On the other hand, if soybean oil is about 25% OA, then 2% Intralipid, which did not stimulate apoB secretion, would have delivered about 1 mg of OA to the liver. Thus, our contention that albumin-bound OA was preferentially linked to the apoB secretory pathway seems strong. We also agree that the roles of the glycerol and phospholipids provided by infusion of Intralipid need to be defined. However, examination of the composition of 20% Intralipid suggests that neither the phospholipid component, of which about 2.5 mg would have been delivered to the liver, or the glycerol content, of which about 5-6 mg would have been delivered to the liver, would have a significant impact relative to the 43 mg of FA delivered over 6 h. Furthermore, 2% Intralipid, which delivered 0.25 mg of phospholipid, 0.5 mg of glycerol, and 4.3 mg of FA to the liver did not stimulate apoB secretion, whereas 0.75 mg of OA did.

The potential role of insulin action, or resistance, in our studies also needs to be addressed in more detail in future studies. Insulin inhibits VLDL secretion acutely in cultured hepatocytes (62, 63) and in normal humans (62, 64, 65), despite increases in TG synthesis. However, insulin-resistant rodents (66) and humans (64, 67) do not exhibit this insulin-induced inhibition of apoB secretion. Our 6-h infusions of 6 mM OA or 20% Intralipid might have produced an insulin-resistant state (48), and this could have contributed to the increased apoB secretion we observed. Neither OA nor Intralipid infusions, however, caused significant changes in plasma insulin or glucose levels. Although we did not determine directly whether increased FA flux caused either global or hepatic insulin resistance, we believe that it is unlikely that the observed effects of either OA or Intralipid infusion on apoB-lipoprotein secretion were mediated by the development of hepatic insulin resistance.

Finally, a preliminary observation that deserves comment was that MTP mRNA was reduced in livers from mice infused with OA for 6 h. A critical player in the early post-transcriptional regulation of apoB secretion (68, 69), MTP appears to function in VLDL assembly by catalyzing both the initiation of lipidation of the nascent apoB polypeptide as well as the formation of triglyceride-rich droplets in the smooth endoplasmic reticulum. These droplets may then fuse with nascent apoB particles to produce mature lipoproteins. Hepatic MTP gene expression can be up-regulated by both high fat (70) and high sucrose (71) diets in hamsters. Ob/Ob mice have increased levels of MTP mRNA and activity (72). In each of these conditions hepatic steatosis and elevated rates of VLDL secretion are present, suggesting that the increases in MTP gene expression are part of a response to maintain hepatic lipid homeostasis. This hypothesis is supported by our finding that hepatic MTP mRNA levels were lower in mice infused with OA for 6 h, a condition where increased apoB secretion in the absence of adequate TG availability led to reductions in hepatic TG content. By contrast, we did not see changes in MTP mRNA after infusion of OA for only 3 h, when apoB secretion had not yet increased, or with Intralipid infusions for either 3 or 6 h, where there were modest increases in both hepatic TG content and secretion. Because MTP protein has a long half-life (54), we do not believe that the changes in mRNA had any effect on the outcomes we observed. The molecular basis of the reduction in MTP gene expression to OA, which would over time be expected to reduce cellular MTP protein and activity, will require further study.

In conclusion, increased FA flux to the liver in vivo can increase secretion of apoB-lipoproteins through post-transcriptional mechanisms. Infusion of a very small quantity of albumin-bound OA stimulated apoB secretion without concomitant stimulation of TG; this uncoupling between increased apoB secretion and the availability of additional TG for co-secretion was associated with reduced hepatic TG mass and decreased hepatic MTP mRNA levels. Studies with a wide range of concentrations of Intralipid indicated that the route of delivery of FA to the liver may be critical for its effects on apoB secretion and supported unique effects for FA and TG on the assembly and secretion of apoB-lipoproteins. Our results, based on in vivo perturbations, could be relevant to the observations that secretion of apoB-lipoproteins are increased in individuals with insulin resistance or with combined hyperlipidemia, a syndrome that has been linked to increased plasma FA flux, whether or not they have hypertriglyceridemia (29-33).

	FOOTNOTES

 
^* This work was supported by NHLBI, National Institutes of Health Grants HL 55368 (to H. N. G.) and HL 62583 (to L.-S. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Visiting scholar from the Chinese Academy of Medical Sciences at Peking Union Medical College Hospital.

^¶ To whom correspondence should be addressed: Dept. of Medicine, PH 10-305, Columbia University College of Physicians and Surgeons, 630 West 168th St., New York, NY 10032; E-mail: hng1{at}columbia.edu.

¹ The abbreviations used are: VLDL, very low density lipoprotein; FA, fatty acid(s); OA, oleic acid(s); TG, triglyceride(s); MTP, microsomal triglyceride transfer protein.

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TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

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