Cellular Aspects of Intestinal Lipoprotein Assembly in Psammomys Obesus: A Model of Insulin Resistance and Type 2 Diabetes

Psammomys obesus gerbils (2.5–3.5 months old) from the Hebrew University colonies were obtained from Harlan (Jerusalem, Israel). After weaning, Psammomys obesus animals were maintained on a low-energy diet containing 2.38 kcal/g (Koffolk, Petach Tikva, Israel) until the beginning of the experiments. They were then switched to a high-energy diet (2.93 kcal/g; Weizmann Institute of Science, Rehovot, Israel) for 2 weeks. The Psammomys obesus gerbils were housed in individual polypropylene cages in a temperature-controlled room with a 12-h light-dark cycle. Water and food were supplied ad libitum. Animals were classified into three groups (A, B, and C) according to plasma glucose and insulin concentrations. All experimental procedures performed in the study were authorized by the Institutional Animal Care Committee.

The jejunum from Psammomys obesus was cleared of mesentery, split longitudinally, washed in culture medium, and cut into explants (3 × 7 mm). Five to seven explants were randomly transferred onto lens paper, with the mucosal side facing up in each organ culture dish (Falcon Plastics, Los Angeles, CA). Six dishes were used for each experimental condition. An amount of medium (0.8 ml) sufficient to dampen the lens paper was added. Explants were cultured in serum-free Leibovitz L-15 medium according to the technique described previously (10,11). After a 30-min stabilization period, the medium was replaced with fresh medium containing a final amount of 1.0 μmol/ml unlabeled oleic acid with 0.5 μCi of [¹⁴C]-oleic acid (specific activity, 53.9 mCi/mmol; Amersham Pharmacia Biotech) in a micellar mixture (6.6 mmol/l sodium taurocholate, 1 mmol/l oleic acid, 0.5 mmol/l monoolein, 0.1 mmol/l cholesterol, and 0.6 mmol/l phosphatidylcholine) (12). Intestinal explants from Psammomys obesus were cultured for 3 h. After this incubation period, tissue integrity was confirmed by morphological (lighted electron microscopy) and biochemical (sucrase activity) studies.

To examine whether higher chylomicron secretion contributed to enhanced in vivo lipemia in insulin-resistant and diabetic Psammomys obesus, a volume of 2 ml of 2% intralipid was orally administered by gavage in 12-h fasted animals. One hour later, Triton WR-1339 (400 mg/kg body wt) was injected. Arterial blood samples were collected 30, 60, and 90 min after Triton administration. Chylomicrons were isolated by ultracentrifugation as described previously (10,11). TGs were quantified enzymatically, and apo B-48 mass was assessed by analytical SDS-PAGE concomitantly with serial dilutions of internal standard apo B-48. The intensity of staining was quantitated by densitometric scanning. Apo B-48 data were extrapolated from the linear standard curve. TG and apo B-48 secretion rates were performed by multiplying the slope of the concentration increase of TGs (in micromoles per milliliter per minute) and apo B-48 (in micrograms per milliliter per minute) over time by the intravascular distribution volume estimated as 4% of body weight.

Aliquots of explant homogenates and their respective incubation media were lipid-extracted with 2:1 (vol/vol) chloroform-methanol (10–12,13). Small amounts of lipid standards were added to the samples before the separation of individual lipid classes by one-dimensional thin-layer chromatography (TLC) (silica gel from Eastman Kodak, Rochester, NY) as described previously (10,12). The nonpolar solvent system was 80:20:3 (vol/vol/vol) hexane-diethylether-glacial acetic acid. The radioactivity of the separated fractions was measured in a Beckman liquid scintillation spectrometer. Quenching was corrected using computerized curves generated with external standards. An aliquot of the tissue homogenate was used for protein determinations (10–13). For the determination of secreted lipoproteins, the medium supplemented with anti-proteases was first mixed with a plasma lipid carrier (2:0.6 vol/vol) to efficiently isolate the de novo TG-rich lipoproteins synthesized. The latter were then isolated at a density of 1.006 g/ml by spinning at 100,000g for 2.26 h with a tabletop ultracentrifuge (Beckman Instruments, Montreal, PQ, Canada) as described previously (10,11).

To provide a carrier for lipoproteins synthesized in vitro, postprandial plasma was obtained from healthy volunteers 3 h after the ingestion of fat (50 g/1.73 m²) as described previously (10,11).

After incubation with 300 μCi [³⁵S]methionine, jejunal explants were washed (three times) with methionine-free Leibovitz medium and homogenized in PBS containing 1% (wt/vol) Triton X-100, methionine (2 mmol/l), phenylmethylsulfonyl fluoride (1 mmol/l), and benzamidine (1 mmol/l). The homogenates were centrifuged (4°C) at 105,000g for 60 min and supernatants subsequently reacted with excess apo B polyclonal antibodies for 18 h at 4°C. Anti-Psammomys obesus apo B antiserum was raised in rabbits. Pansorbin was then added, and the mixture was reincubated at 20°C for 60 min. The immunoprecipitates were washed extensively and analyzed by a linear 4–20% acrylamide gradient preceded by a 3% stacking gel as described previously (13). Apo B-48 bands on gels were sectioned and counted after an overnight incubation at 20°C with 1 ml BTS-450 (Beckman) and 10 ml liquid scintillation fluid (Ready Sol. NA, Beckman).

Intestinal microsomes used as the source of microsomal triglyceride transfer protein (MTP) activity were isolated as described previously (14). MTP activity was determined by the transfer of radiolabeled triacylglycerol from donor small unilamellar vesicles (40 nmol egg phosphatidylcholine, 0.08 nmol [¹⁴C]triacylglycerol, and 2 nmol cardiolipin) to acceptor small unilamellar vesicles (240 nmol egg phosphatidylcholine and 0.48 nmol triacylglycerol) at 37°C for 1 h. This assay was previously described in detail (14). Lipid transfer activity is expressed as a percentage of TG transfer per unit time under verified linear assay conditions (first-order kinetics). To assess the presence of MTP and evaluate its mass, homogenates of intestinal tissue were prepared for Western blotting as described previously (14). Proteins were separated on a 4–20% gradient SDS-PAGE and electroblotted onto nitrocellulose membranes. Nonspecific binding sites of the membranes were blocked using defatted milk proteins followed by the addition of primary antibodies directed against MTP. The relative amount of primary antibody was detected with horseradish peroxidase-conjugated secondary antibody. Blots were developed, and the mass of MTP was quantitated using an HP Scanjet scanner equipped with a transparency adapter and software.

Activities of monoacylglycerol acyltransferase (MGAT) and diacylglycerol acyltransferase (DGAT) were determined in microsomes as reported by Coleman (15).

Jejunal explants were homogenized and immunoprecipitated with anti-I-fatty acid binding protein (FABP) and anti-L-FABP antibodies raised in rabbits after the injection of recombinant human I- and L-FABP. The immunoprecipitates were run on SDS-PAGE and transferred to nitrocellulose membrane, which was blotted with I-FABP and L-FABP antibodies. Control experiments were performed to confirm the specificity of the antibodies. Quantitation of I- and L-FABP was carried out as described above.

Plasma glucose was determined by an enzymatic glucose analyzer, and insulin levels were assessed by radioimmunoassay using a human primary antibody (Phadesph; Kabi Pharmacia Diagnostics, Uppsala, Sweden). Plasma TG and cholesterol levels were measured colorometrically (Boehringer Mannheim, Mannheim, Germany).

Data were statistically analyzed by one-way ANOVA to assess differences in the parameters studied. Differences between mean values were evaluated by Student’s two-tailed t test.

As expected, genetically predisposed Psammomys obesus on an affluent laboratory rodent diet higher in energy than the desert native food displayed the three characteristic patterns of normoinsulinemia/normoglycemia (group A), hyperinsulinemia/normoglycemia (group B), and hyperinsulinemia/hyperglycemia (group C) (Fig. 1). Furthermore, the latter two animal groups exhibited moderate weight gain (Fig. 1) as well as hypertriglyceridemia and hypercholesterolemia (Fig. 2), despite food intake comparable to controls (∼15 g/day).

The profile of TG and cholesterol in intestinal tissue was determined in Psammomys obesus animals. Lipid analysis revealed higher levels of TGs in groups B and C than in group A (Fig. 3). Although cholesterol fractions were also increased in groups B and C, statistical significance was reached only for total cholesterol and free cholesterol in group C compared with group A.

We measured the activity of MGAT and DGAT to evaluate the effect of insulin resistance and diabetes on the major enzymatic machinery involved in triacylglycerol formation in the intestine. As observed in Fig. 4, MGAT and DGAT activity was elevated in groups B and C compared with group A. Accordingly, the esterification of [¹⁴C]oleic acid into complex lipid fractions in jejunal explants of groups B and C exceeded that of group A (Fig. 5A). Likewise, the release of labeled TGs, phospholipids, and cholesteryl esters into the medium was higher in group B and C than in group A animals (Fig. 5B). The current findings suggest that insulin resistance and diabetes enhanced the intestine’s ability to produce lipids in Psammomys obesus.

The synthesis of apo B was determined in jejunal explants in culture (Fig. 6). As evidenced by [³⁵S]methionine incorporation, apo B-48 synthesis was significantly increased in the intestine of groups B and C relative to that of group A (P < 0.02). With labeled oleic acid used as a substrate, our data revealed significantly (P < 0.01) more output of TG-rich lipoproteins in groups B and C than in group A (Fig. 7). However, with the addition of the proteasomal inhibitors (N-acetyl-leucyl-norleucinat and lactacystin) to the culture medium, the differences in de novo apo B synthesis in groups A, B, and C disappear. Overall, our observations suggest that the development of insulin resistance and diabetes in Psammomys obesus stimulated the intestinal transport of lipids by augmenting apo B-48 synthesis through reduced proteasomal degradation activity, resulting in enhanced TG-rich lipoprotein assembly and secretion.

Because MTP is necessary for the efficient assembly of TG-rich lipoproteins, we assessed MTP activity and protein expression in the intestine of Psammomys obesus. There were no differences in MTP activity and protein expression among groups A, B, and C (Fig. 8). Our results, therefore, suggest that MTP does not play a role in the intestinal overproduction of the TG-rich lipoprotein observed in insulin-resistant and diabetic Psammomys obesus animals.

The next set of experiments was designed to examine the protein expression of the two distinct FABPs, I- and L-FABP, in the intestine in the various Psammomys obesus groups. The effect of insulin resistance or diabetes was noted on the abundance of I-FABP, whereas levels of L-FABP were higher in groups B and C than in group A (Fig. 9). This suggests a relationship between the magnitude of L-FABP and the increase in intestinal lipid transport in insulin resistance and diabetes.

To confirm intestinal overproduction in insulin-resistant and diabetic animals, we determined the effect of dietary fat on the increase in chylomicron TG and apo B-48 moieties after the injection of Triton WR-1339. In individual animals, chylomicron-TG and chylomicron-apo B-48 rose linearly with time, irrespective of group, with no tendency to plateau at the time Triton was administered and for the subsequent 90 min (data not shown). Production rates were derived from the slope of the increase over 90 min. After the Triton WR-1339 lipolysis blockade, the levels of chylomicron-TG and chylomicron-apo B-48 were higher in insulin-resistant and diabetic animals compared with normal Psammomys obesus (Fig. 10) animals, indicating the efficiency of these abnormal conditions to stimulate in vivo fat transport.

Hypertriglyceridemia is very common in diabetes and substantially increases the risk of cardiovascular diseases. Similarly, insulin resistance is associated with elevated plasma TGs and predisposes for cardiovascular complications. Although the regulation of circulating TG-rich lipoproteins is extremely complex in diabetes and insulin resistance, many studies consider increased hepatic VLDL secretion and impaired VLDL clearance mechanisms for dyslipidemia (5,6). It is postulated that the overall effect of insulin resistance and diabetes on VLDL production reflects raised fatty acid flux to the liver (6). Consequently, there is a stimulation of the assembly and output of apo B-100-containing lipoproteins (6,16). The aim of our investigation was to examine whether intestinal TG metabolism is also altered in Psammomys obesus animals that are characterized by insulin resistance and diabetes (17,18). We demonstrated that the development of the insulin-resistant/diabetic state is accompanied by 1) an intestinal increase in lipid content and de novo TG synthesis concomitant with an increased amount of L-FABP and decreased levels of I-FABP; 2) augmented MGAT and DGAT activity, two enzymes that belong to the primary triacylglycerol synthesis pathway in enterocytes; 3) elevated apo B-48 biogenesis, probably resulting from reduced proteasomal degradation; 4) stimulated TG-rich lipoprotein assembly; and 5) unaltered MTP activity and protein expression.

Triacylglycerol biosynthesis in the intestine is believed to occur mainly through the monoacylglycerol pathway. Initially, monoacylglycerol and fatty acyl CoA are covalently joined to form diacylglycerol in a reaction catalyzed by MGAT (19,20). Then, diacylglycerol and fatty acyl CoA are used to synthesize triacylglycerol in a reaction catalyzed by DGAT (19,20). In our study, measurement of MGAT and DGAT disclosed a high activity level, which is consistent with the remarkable capacity of the small intestine to produce triacylglycerol in an insulin-resistant and diabetic state. Physiologically, the present findings reflect the intestine’s attempt to enhance lipid production under insulin resistance and diabetic conditions. It is generally accepted that the intracellular mechanism of TG-rich lipoprotein assembly requires apo B synthesis and association. The early step in this process is the co-translational lipidation of apo B that is transiently bound to the endoplasmic reticulum membrane where it is folded. The addition of lipids stabilizes apo B and prevents its proteolytic degradation via the ubiquitin-dependent proteasomal pathway (21,22). It is, therefore, reasonable to suggest that the augmented TG synthesis in the small intestine of Psammomys obesus insulin-resistant and diabetic animals enhanced apo B-48 protection from misfolding and degradation, resulting in the marked secretion of apo B-48-containing TG-rich lipoproteins. Our data argue for an increased efficiency of intestinal TG-rich lipoprotein assembly and secretion and resemble recent observations in fructose-fed hamsters exhibiting chronic hyperinsulinemia and insulin resistance while displaying a significant stability enhancement of newly synthesized apo B with only a minor fraction being sorted to intracellular degradation (10).

MTP is a resident protein in the lumen of the endoplasmic reticulum that facilitates the transfer of lipids from their site of synthesis in the endoplasmic reticulum membrane into the lumen during the assembly of TG-rich lipoproteins (23,24). However, the MTP requirement for the export of apo B-48 remains controversial. Recent studies have stressed that liver-specific MTP knockout mice still secrete apo B-48, thus excluding the obligatory role of MTP (25,26). Our findings show that Psammomys obesus enterocytes are capable of releasing more TG-rich lipoproteins in insulin-resistant and diabetic states without augmenting MTP activity or protein expression. Similar data were obtained in genetically diabetic obese rats (27). Conversely, Haidari et al. (9) suggested that MTP may play an important role in the postprandial dyslipidemia in insulin-resistant hamster and Zucker obese fa/fa, respectively (28). Although this discrepancy may be animal species-dependent, additional work is necessary to delineate the role of MTP in apo B-48 delivery, particularly in insulinemic and diabetic states.

FABPs are another set of proteins critical to the transport of fatty acids in the enterocyte (28). Based on a variety of physico-chemical studies, different functions have been proposed for cytosolic FABPs, including fatty acid transport and compartmentalization, the modulation of enzyme activities involved in lipid metabolism, and the protection of cellular integrity from the detrimental effects of hydrophobic fatty acid or other noxious substances (28–30). The physiological significance of the presence of the two FABP forms (L- and I-FABP) in intestinal absorptive cells remains unknown. Present data show a relationship between endogenous L-FABP and TG-rich lipoprotein secretion, both of which are induced in the small intestine of insulin-resistant and diabetic Psammomys obesus. On the other hand, I-FABP protein expression appears to be decreased and uncoupled with the stimulated intestinal lipid transport in insulin-resistant and diabetic Psammomys obesus animals. Accordingly, recent studies in mice have suggested that I-FABP is not essential for dietary fat absorption (31), and its overexpression may even limit intestinal lipid transport, as noted in Caco-2 cells (32). On the basis of these observations, we suggest that insulin-resistant and diabetic conditions may induce L-FABP that may be involved in the specific targeting of fatty acid to the synthetic pathway, thereby stimulating lipid esterification and TG-rich lipoprotein assembly. It is conceivable that the induction of L-FABP results in more efficient conveyance of fatty acids to the endoplasmic reticulum, which preserves apo B-48 from proteolytic degradation and enhanced chylomicron formation.

Hypertriglyceridemia frequently occurs in individuals with type 2 diabetes and contributes to vascular complications. More and more studies have emphasized the influence of postprandial TG-rich lipoproteins on the acceleration of atherosclerosis and the progression of coronary artery disease (7,33). In the current report, we have shown that the standard diet administered to Psammomys obesus, a well-defined model for dietary-induced insulin resistance and type 2 diabetes, provoked increased enterocyte lipid content as well as higher efficiency in intestinal de novo TG synthesis, apo B-48 biogenesis, and chylomicron production. Many differences were noted in these processes between groups B and C and may be due to the degree of insulin resistance and/or hyperglycemia per se. Hyperglycemia is a significant pathophysiological state in diabetes that has been suggested to play a key role in the pathogenesis of vascular dysfunction in diabetes via different mechanisms, such as protein glycosylation, sorbitol accumulation, protein kinase increase, oxidative stress, glycotoxicity, and others (34,35). Further studies of this model may provide additional insight into the separate role of insulin resistance and hyperglycemia in the development of these abnormal intestinal fat processes, which may possibly help identify novel approaches to prevention or treatment.

In conclusion, evidence from the Psammomys obesus insulin-resistant and diabetic model indicates that intestinal TG-rich lipoprotein overproduction can contribute to the development of hypertriglyceridemia. A number of intracellular factors have been found to be associated with the deregulatory mechanisms, including de novo lipid synthesis, the monoacylglycerol pathway, L-FABP mass, reduced proteasomal degradation of apo B, and lipoprotein assembly. Further understanding of the physiopathology of intestinal lipid transport in insulin resistance and diabetes should facilitate the design of new therapeutic strategies for the prevention and treatment of hyperlipidemia and atherosclerosis.

This study was supported by research grants from the Canadian Institutes of Health Research (MT-10583) and by research scholarship awards from the Fonds de la Recherche en santé du Québec, awarded to E.S. and E.L.

We acknowledge the expert secretarial assistance of Schohraya Spahis.