Increased Intestinal Lipid Absorption Caused by Ire1β Deficiency Contributes to Hyperlipidemia and Atherosclerosis in Apolipoprotein E–Deficient MiceNovelty and Significance
Rationale: High fasting serum lipid levels are significant risk factors for atherosclerosis. However, the contributions of postprandial excursions in serum lipoproteins to atherogenesis are less well-characterized.
Objective: This study aims to delineate whether changes in intestinal lipid absorption associated with loss of inositol-requiring enzyme 1β (Ire1β) would affect the development of hyperlipidemia and atherosclerosis in Apoe−/− mice.
Methods and Results: We used Ire1β-deficient mice to assess the contribution of intestinal lipid absorption to atherosclerosis. Here, we show that Ire1b−/−/Apoe−/− mice contain higher levels of intestinal microsomal triglyceride transfer protein, absorb more lipids, exhibit hyperlipidemia, and have higher levels of atherosclerotic plaques compared with Apoe−/− mice when fed chow and western diets.
Conclusions: These studies indicate that Ire1β regulates intestinal lipid absorption and that increased intestinal lipoprotein production contributes to atherosclerosis.
Excess plasma lipids are risk factors for various cardiovascular and metabolic diseases.1 The relationship between excess plasma lipids and cardiovascular disease is based on lipid measurements made in the fasting state. This implies that fasting lipemia attributable to increased hepatic lipoprotein production or delayed catabolism of hepatic-derived lipoproteins drives the increased cardiovascular risk. However, humans spend much of the day in a postprandial (fed) state, and it has been proposed that chylomicron remnants arising during the postprandial state also contribute to atherosclerosis because these particles are taken-up by arterial smooth muscle cells.2–4 A role for absorbed lipids in atherosclerosis is supported by the association of atherosclerosis with delayed elimination of postprandial triglyceride-rich lipoproteins,5 the presence of apoB48 in the atherosclerotic plaque,6 uptake and retention of chylomicron remnants in the arterial wall,7 and increased inflammatory response to postprandial lipoproteins.8 A problem associated with establishing a relationship between intestinal lipoproteins and atherosclerosis is the presence of and associated changes in hepatic lipoproteins in the postprandial state. For example, it is well-known that absorption of lipids during postprandial state is also associated with increased secretion of hepatic lipoproteins.5 Veniant et al9 showed that postprandial atherogenicity might be correlated with apoB48-contaning lipoproteins. However, comparison of atherosclerosis in apoB48 and apoB100 only mice in Apoe−/− background indicated that atherosclerosis was more likely related to plasma cholesterol concentration and was independent of the apoB molecules.9
To address this issue, we took advantage of our previous observations that inositol-requiring enzyme 1β (Ire1β) is only expressed in the intestine10–12 and that it suppresses intestinal lipid absorption by regulating microsomal triglyceride transfer protein (MTP),13,14 a critical chaperone required for the assembly and secretion of apoB-containing lipoproteins.15–17 Mechanistic studies have shown that Ire1β decreases MTP mRNA involving posttranscriptional degradation.13 These studies also showed that hyperlipidemia observed in Ire1b−/− mice fed a high-cholesterol and high-fat diet is attributable to increased intestinal lipid absorption with little contribution from hepatic lipoprotein production. However, it was not clear from these studies whether this hyperlipidemia represents a response to high dietary cholesterol and fat or if diets rich in high cholesterol and fat accelerate development of this phenotype. Another question that remained unanswered was whether changes in intestinal lipid absorption would affect atherosclerosis. Here, we address the role of the selective increase in intestinal lipid absorption associated with loss of Ire1β in the development of hyperlipidemia and atherosclerosis in Apoe−/− mice.
[14C]cholesterol, [3H]cholesterol, [3H]oleic acid, and [3H]triolein were from NEN LifeScience Products. Oleic acid, cholate, deoxycholate, taurocholate, and monoacylglycerol were procured from Sigma. Phosphatidylcholine was from Avanti Polar Lipids. Other chemicals and solvents were purchased from Fisher Scientific.
Mice and Diets
The apoE-deficient (Apoe−/−) mice on a C57Bl/6J background were purchased from Jackson Laboratory (Bar Harbor, ME). Ire1b−/− mice,11 originally of a 129svev background, were backcrossed with C57Bl/6J Apoe−/− mice for eight generations to generate Ire1b−/−/Apoe−/− mice. Mice were housed in an air-conditioned room at 22°C±0.5°C with a 12-hour lighting schedule (700–1900 hours). Mice were kept on a chow diet (Research Diets) or fed a western-type diet (0.15% cholesterol, 20% saturated fat; Research Diets). Only male mice were used in these experiments to avoid the effects of hormonal changes on plasma lipids. Experiments involving animals were conducted with the approval of State University of New York Downstate Medical Center Institutional Animal Care and Use Committee.
Plasma Lipid and Cytokine Measurements
Total cholesterol and triglyceride levels were measured using kits (Thermo Trace, Melbourne, Australia). The high-density lipoprotein lipids were measured after precipitating apoB lipoproteins.18 Plasma cytokines were detected using the Mouse Common Cytokines Multi-Analyte ELISArray Kit from SABiosciences according to the manufacturer's guidelines.
Plasma lipoproteins were isolated by gel filtration (flow rate of 0.2 mL/min, 200-μL fractions, 10 mmol/L Tris, pH 7.4 buffer) using a Superose 6 column. The very-low-density lipoprotein/low-density lipoprotein fractions were pooled and their percent compositions were determined. Further, they were subjected to negative staining and electron microscopy.19 Lipoproteins (density<1.006 g/mL) were also isolated by ultracentrifugation (Beckman Coulter Optima Tabletop Ultracentrifuge, TLA-100 rotor, 100,000 rpm, 180 minutes, 4°C) after overlaying 150 μL of plasma with 6 mL of <1.006 g/mL solution. The top (200 μL) was collected and used for composition analysis and negative staining.
Postprandial Lipid Absorption
For postprandial lipid measurements, plasma samples were collected at 22:00 hours (3 hours after lights were turned off). For daytime restricted feeding experiments, mice had access to food only from 9:30 am to 1:30 pm for 10 days. On the last day, blood was withdrawn at hourly intervals for 6 hours starting at 9:30 am (just before the food was given). Total cholesterol and triglyceride levels were measured in the plasma as described.
Hepatic Lipid Mobilization and Metabolic Studies
Hepatic triglyceride secretion rates were determined in vivo.20 Mice were fasted for 16 hours and injected intraperitoneally with poloxamer 407 (30 mg/mouse). Blood samples were drawn before injection and every 30 minutes for 3 hours for triglyceride determinations. In a separate experiment, mice were fasted for 16 hours and intraperitoneally administered with [35S]-Promix (Perkin Elmer) along with poloxamer 407 (30 mg/mouse). To study hepatic apoB secretion, apoB-containing lipoproteins were immunoprecipitated from the plasma, separated on gel, dried, and fluorographed. Albumin was used as a control.
Short-Term Lipid Absorption Studies
Age-matched male mice (four per group) fed a chow diet for 1 year or western diet for 6 weeks were fasted overnight and injected intraperitoneally with poloxamer 407 (30 mg/mouse). After 1 hour, mice were gavaged with 0.5 μCi of either [3H]triolein or [14C]cholesterol with 0.2 mg of unlabeled cholesterol in 15 μL of olive oil.21 After 2 hours, plasma was used to measure radioactivity.
Uptake and Secretion of Lipids by Primary Enterocytes
To study uptake, primary enterocytes from overnight fasted Apoe−/− and Ire1b−/−/Apoe−/− mice (n=3) were suspended in 4 mL of DMEM containing 0.5 μCi/mL of [3H]oleic acid or [3H]cholesterol and incubated at 37°C.18,21 After 1 hour, enterocytes were washed and lipids were isolated to determine uptake of radiolabeled lipids. For characterization of secreted lipoproteins, enterocytes were isolated from overnight fasted mice and labeled for 1 hour with 0.5 μCi/mL of [3H]oleic acid or [3H]cholesterol. Enterocytes were washed and incubated with fresh media containing 1.4 mmol/L oleic acid–containing micelles.18,22 After 2 hours, enterocytes were centrifuged and supernatants were collected. In [3H]oleic acid labeling experiment, lipids were isolated from the media and separated on thin-layer chromatography to quantify incorporation of [3H]oleic acid into triglycerides and phospholipids. In [3H]cholesterol labeling experiment, media was subjected to density gradient ultracentrifugation to determine its secretion with different lipoproteins.18
Determination of MTP Activity and mRNA Levels
Small pieces (0.1 g) of liver and proximal small intestine (approximately 1 cm) were homogenized in low-salt buffer and centrifuged, and supernatants were used for protein determination and the MTP assay23,24 using a kit (Chylos). Total RNA from tissues was isolated using TriZol (Invitrogen) and MTP mRNA levels were determined.13
Mouse Atherosclerotic Lesion Measurement
The aortas were dissected and the arches were photographed.25 Exposed aortas were stained with Oil Red O and en face assay was performed.25 The heart was fixed in 4% formaldehyde and then embedded in paraffin. The aortic root was sectioned into 7-μm-thick slices and then stained with Harris hematoxylin and eosin. Histological and morphological analyses of collagen were performed after Masson trichrome staining (Richard-Allan Scientific) of these sections. Images were viewed and captured with a Nikon Labophot 2 microscope equipped with a SPOT RT3 color video camera attached to a computerized imaging system. The average area of these aortic root lesions from each animal was quantified using Image–Pro Plus version 4.5 (Media Cybernetics).
Ablation of IRE1β in Chow-Fed Apoe−/− Mice Increases Plasma Lipids
We have shown previously that Ire1b affects lipid absorption during high-cholesterol or western diet feeding.13 We wondered whether IRE1β knockout mice will have development of hyperlipidemia on apoE knockout background when fed a chow diet. Another question we asked was whether these mice will be more susceptible to atherosclerosis. To evaluate the role of IRE1β on hyperlipidemia and atherosclerosis, we crossed Ire1b−/− mice with Apoe−/− mice to generate Ire1b−/−/Apoe−/− mice. There was no significant change in the body weight of these mice after 12 months of chow diet (Table 1). Furthermore, these mice did not register any significant differences in fasting plasma glucose, phospholipids, and free fatty acid levels (Table 1). However, Ire1b−/−/ Apoe−/− mice showed a significant increase of 56% and 40% in fasting plasma triglyceride and cholesterol (Table 1), respectively, compared with Apoe−/− mice, mainly because of augmentations in apoB-containing lipoproteins (Supplemental Figure IA, B). Further, plasma of Ire1b−/−/Apoe−/− mice had 43% more proatherogenic apoB48 levels compared with Apoe−/− mice (Supplemental Figure IC, D). Negative staining and electron microscopy (Supplemental Figure IE, Apoe−/−; Supplemental Figure IF, Ire1b−/−/Apoe−/−) showed an increase in particles with diameters of 80 to 140 nm in Ire1b−/−/Apoe−/− mice (Supplemental Figure IG), but compositional analysis did not reveal any differences between the two groups (Supplemental Figure IH). Characterization of plasma lipoproteins was also performed after isolating density<1.006 g/mL lipoproteins by ultracentrifugation (Figure 1A, Apoe−/−;Figure 1B, Ire1b−/−/Apoe−/−). Again, there were more particles with diameters ranging between 80 and 140 nm (Figure 1C), with no significant differences in the composition of these particles (Figure 1D). Western blot analysis revealed approximately 3.3-fold increase in apoB48 levels (Figure 1E, F). These data indicate that Ire1β deficiency augments hyperlipidemia in Apoe−/− mice by increasing number of apoB-containing remnant lipoproteins with no significant effect on their composition.
Hyperlipidemia Attributable to Increased Lipid Absorption in Chow-Fed Ire1b−/−/Apoe−/− Mice
Next, we attempted to elucidate mechanisms for the origin of apoB lipoproteins and hyperlipidemia in these mice. Increases in plasma apoB lipoproteins in Apoe−/− mice that are deficient in clearing apoB lipoproteins could be explained by their enhanced assembly and secretion by the liver or intestine or both. First, we determined hepatic secretion of triglycerides in these mice. Overnight fasted animals were injected with lipoprotein lipase inhibitor poloxamer P407 and 35S-Promix, and plasma lipids and nascent apolipoproteins were determined. There were no differences in time-dependent increases in plasma triglycerides (Figure 2A), and triglyceride production rates were similar between Ire1b−/−/Apoe−/− (188.7±16.7 mg/dL/h) and Apoe−/− (191.2±30.6 mg/dL/h) mice (Figure 2B). Moreover, these mice had similar plasma levels of nascent apoB48, apoB100, and albumin (Figure 2C, D). Thus, differences in plasma lipids are not related to changes in hepatic lipoprotein production.
Second, we looked at the absorption of lipids by the intestine. Mice fed chow diet for 12 months were injected intraperitoneally with a lipoprotein lipase inhibitor poloxamer P407 and were fed with radioactive cholesterol and triolein in olive oil. There was a significant increase in the appearance of [3H]triolein and [14C]cholesterol in the plasma of Ire1b−/−/ Apoe−/− versus Apoe−/− (Table 2). Gel filtration analysis showed increased lipid counts in the apoB lipoproteins of Ire1b−/−/Apoe−/− mice (Figure 2E, F). Additionally, total mass of triglyceride and of cholesterol were increased (Table 2) in apoB lipoproteins (Figure 2G, H). These studies indicate that Ire1b−/−/Apoe−/− mice absorb more lipids via apoB lipoprotein production.
Increased intestinal lipid absorption could be attributable to increased lipid uptake by enterocytes or their subsequent enhanced secretion by these cells. To differentiate between these two steps, we assessed the amounts of radioactivity present in the proximal intestine (approximately 10 cm long) of these mice during short-term absorption studies. There was no significant difference in the levels of intestinal triolein and cholesterol counts between Ire1b−/−/Apoe−/− and Apoe−/− mice (Table 2). We also used isolated primary enterocytes to explore mechanisms for enhanced lipid absorption. First, we studied the uptake of oleic acid and cholesterol and found no differences in the uptake (Table 2). To substantiate the observation that oleic acid and cholesterol uptake was not affected, we also measured mRNA levels of candidate transporters. Analysis of the mRNA levels of CD36, SR-B1, NPC1L1, ABCG5, and ABCG8 in the intestine of chow diet–fed mice did not register any significant difference between Ire1b−/−/ Apoe−/− and Apoe−/− mice (Figure 2I). Furthermore, we did not see any change in the levels of CD36 protein (Figure 2I, inset). Hence, Ire1β deficiency does not appear to affect oleic acid or cholesterol uptake by enterocytes.
Next, we studied secretion of oleic acid–labeled lipids by these enterocytes. Secretion of oleic acid-labeled triglycerides was significantly increased by Ire1b−/−/Apoe−/− enterocytes (Table 2). Similarly, these enterocytes secreted more cholesterol into the media compared with Apoe−/− enterocytes (Table 2). We have previously shown that cholesterol can be secreted as part of chylomicrons and high-density lipoprotein particles.18,21,26 Density gradient ultracentrifugation analysis showed that cholesterol secretion was increased in chylomicron fractions (Figure 2J). These results suggest that Ire1b−/−/ Apoe−/− enterocytes secrete more chylomicrons.
If Ire1b−/−/Apoe−/− mice were making more chylomicrons and absorbing more lipids, then we reasoned that fecal lipid loss might be lower and plasma lipid levels might be higher during postprandial state in the absence of Ire1β. Although it is known that fecal excretion of fat is low, we measured this in feces collected over a 24-hour period and observed that fecal triglyceride and cholesterol levels were significantly lower in Ire1b−/−/Apoe−/− compared with Apoe−/− mice (Table 1). To determine postprandial lipemia, plasma samples were collected at 22:00 hours from Ire1b−/−/ Apoe−/− and Apoe−/− mice. Plasma triglyceride and cholesterol were significantly higher by 70% and 56%, respectively, in Ire1b−/−/Apoe−/− compared with Apoe−/− mice (Table 1). To substantiate further that Ire1β deficiency increases hyperlipidemia at mealtime, we provided food for 4 hours during the day for 10 days.27–29 Plasma analysis during mealtime showed that Ire1b−/−/Apoe−/− mice had higher lipid levels at all times during food availability compared with Apoe−/− mice (Figure 2K, L). These studies indicate that postprandial response in Ire1b−/−/Apoe−/− mice is higher than in Apoe−/− mice. Further, they show that higher lipids persist even after fasting. Therefore, postprandial hyperlipidemic excursions are more significant and sustained in Ire1β-deficient mice.
To understand molecular basis for increases in plasma lipids and lipoproteins, we looked at the expression of apoB and MTP, two proteins critically involved in apoB lipoprotein assembly and secretion. There was a 40% increase and a 47% increase, respectively, in the intestinal MTP activity and mRNA levels compared with the levels in Apoe−/− mice; in contrast, no significant changes in the hepatic MTP activity and mRNA were noted (Table 1). Furthermore, there were no significant differences in apoB mRNA levels in the intestine and liver of these mice (Table 1). These results suggest that ablation of IRE1β in apoE-null background leads to increased expression of intestinal MTP, but not apoB.
Increased Atherosclerosis in Ire1b−/−/Apoe−/− Mice
To evaluate the impact of IRE1β deficiency on atherogenesis, Apoe−/− and Ire1b−/−/Apoe−/− mouse aortas were dissected and photographed after 12 months of chow diet (Figure 3A). Ire1b−/−/Apoe−/− mice had more extensive plaques compared with Apoe−/− mice at the branch points. En face analyses of Oil Red O–stained thoracic aortic areas exhibited marked increases in atherosclerotic lesions in Ire1b−/−/Apoe−/− mice compared with Apoe−/− mice (Figure 3B). Quantification of the stained lesions indicated approximately 50% increased plaque areas in Ire1b−/−/Apoe−/− mice (Figure 3C). Analysis of the plaque morphology in hematoxylin and eosin–stained cross-sections of the cardiac/aortic junctions revealed substantial differences between Ire1b−/−/Apoe−/− and Apoe−/− mice (Figure 3D). Quantification revealed that the proximal aortas of Ire1b−/−/Apoe−/− mice had approximately 43% increased lesion area (Figure 3E). Further, plaques from Ire1b−/−/Apoe−/− mice contained more necrotic core (Figure 3D), but percent necrotic core areas in these lesions were not statistically different (Figure 3F). However, plaques in Ire1b−/−/Apoe−/− mice had substantially less collagen (29%), as illustrated by the analysis of trichrome-stained images (Figure 3G, H). These studies indicate that Ire1b−/−/Apoe−/− mice are more susceptible to atherosclerosis than Apoe−/− mice.
Accelerated Hyperlipidemia and Increased Atherosclerosis in Ire1b−/−/Apoe−/− Mice Fed a Western Diet
Next, we studied the effect of a western diet on plasma lipids. Age-matched (3-month-old males) Ire1b−/−/Apoe−/− and Apoe−/− mice were fed a western diet for 6 weeks. Similar to chow-fed mice, no difference in the body weights was observed between Ire1b−/−/Apoe−/− and Apoe−/− mice fed the western diet (Table 1). Again, no differences in the fasting plasma glucose, phospholipids, and free fatty acid levels were observed between Ire1b−/−/Apoe−/− and Apoe−/− mice (Table 1). However, the western diet increased fasting plasma triglyceride and cholesterol (Table 1) in Ire1b−/−/Apoe−/− mice compared with Apoe−/− mice mainly because of increases in very-low-density lipoprotein/low-density lipoprotein fractions (Supplemental Figure IIA, B). We then studied changes in MTP levels. Double-knockout mice had higher intestinal MTP activity and mRNA levels, but there were no significant differences in the hepatic MTP expression (Table 1). Further, plasma of Ire1b−/−/Apoe−/− mice had 93% more proatherogenic apoB48 levels compared with Apoe−/− mice (Supplemental Figure IIC, D). These studies show that Ire1b−/−/Apoe−/− mice have increased hyperlipidemia when fed a western diet.
Ire1b−/−/Apoe−/− mice had more extensive plaques compared with Apoe−/− mice (Figure 4A) and showed a marked increase (approximately 50%) in atherosclerotic lesions (Figure 4B, C). Analysis of the plaque morphology after hematoxylin and eosin staining (Figure 4D) demonstrated approximately 63% increase in lesion area (Figure 4E) in the proximal aortas of Ire1b−/−/Apoe−/− mice with more necrotic core (Figure 4D), but percent necrotic core areas in these lesions were not statistically different (Figure 4F). Trichrome-stained images showed substantially less collagen (30%) in the plaques of Ire1b−/−/Apoe−/− mice compared with Apoe−/− mice (Figure 4G, H). Analysis of various cytokines and inflammatory markers in the plasma revealed significant increases in IL-1α, IL-6, and IL-10 in Ire1b−/−/Apoe−/− mice (Figure 4I). These studies indicate that Ire1b−/−/ Apoe−/− mice fed a western diet are more susceptible to atherosclerosis than Apoe−/− mice.
Mechanistic studies revealed that western diet–fed Ire1b−/−/ Apoe−/− mice absorbed 55% and 68% more [3H]triolein and [14C]cholesterol, respectively, compared with Apoe−/− mice (Table 2). Gel filtration analysis showed increased counts in the apoB lipoproteins of Ire1b−/−/Apoe−/− mice (Figure 5A, B). There was a 38% increase and a 32% increase in total triglyceride and cholesterol mass, respectively (Table 2), attributable to increases in apoB lipoproteins (Figure 5C, D). These studies indicate that Ire1b−/−/Apoe−/− mice absorb more lipids via apoB lipoprotein production. Next, we studied the uptake of cholesterol by enterocytes isolated from western diet–fed mice and found no differences (Table 2). Analysis of the mRNA levels of CD36, SR-B1, NPC1L1, ABCG5, and ABCG8 in the intestine of western diet–fed mice also did not register any significant difference between Ire1b−/−/Apoe−/− and Apoe−/− mice (Figure 5E). Hence, Ire1β deficiency does not affect cholesterol uptake by enterocytes. However, Ire1b−/−/ Apoe−/− enterocytes secrete more cholesterol into the media compared with Apoe−/− enterocytes (Table 2), and this increase was mainly in chylomicron fractions (Figure 5F).
We previously reported that Ire1β deficiency results in hyperlipidemia in C57Bl/6J mice fed high-cholesterol and western diets.13 Here, we show that Ire1β deficiency enhances hyperlipidemia in 1-year-old Apoe−/− mice fed a chow diet. Increases in plasma lipids were accelerated when Ire1b−/−/Apoe−/− mice were fed a western diet. These studies indicate that Ire1β deficiency increases susceptibility toward hyperlipidemia independent of the genetic background, and this propensity is further augmented when more dietary fat is available.
Physiological studies indicated that a major reason for hyperlipidemia in Ire1b−/−/Apoe−/− mice compared with Apoe−/− mice was increased intestinal lipid absorption with no significant change in hepatic lipoprotein production (Figure 2A–J). Significant hyperlipidemia persisted in these mice after fasting as well as during the day during ad libitum feeding. Moreover, food entrainment studies indicated that hyperlipidemia was more pronounced at the time of food availability (Figure 2K, L). Despite increased absorption, there were no significant differences in body weights of Ire1b−/−/Apoe−/− and Apoe−/− mice, indicating that higher absorption at mealtime results in sustained hyperlipidemia and not in obesity.
Further studies were performed to delineate biochemical mechanisms involved in lipid absorption. These studies showed that Ire1β deficiency has no effect on oleic acid or cholesterol uptake by enterocytes, but these enterocytes secrete more lipids with chylomicrons. It is known that free fatty acids are taken-up by enterocytes and hepatocytes. After uptake, fatty acids are converted to triglycerides that are either incorporated into newly synthesized lipoproteins and secreted or stored in the cytoplasm. It is also known that lipid secretion is a slower process than fat uptake. For this reason, during the postprandial state there is significant accumulation of fat in enterocytes.30 This fat is eventually transported to plasma, but after a significant lag. In human studies, it is now well-established that dietary lipids ingested during one meal are present in chylomicrons secreted during the next meal.31,32 Our studies suggest that Ire1β does not affect lipid uptake, but it helps in faster intracellular processing and secretion of lipids with intestinal lipoproteins.
We had previously shown that one molecular mechanism for increased intestinal lipid absorption was enhanced MTP expression.13 Again, in Ire1b−/−/Apoe−/− mice we observed significant augmentations in MTP mRNA and activity with no alterations in hepatic MTP levels. Therefore, it is likely that increases in intestinal MTP expression contribute to increased chylomicron assembly and lipid absorption in the absence of Ire1β. This leads to sustained hyperlipidemia in the absence of apoE.
It is unclear why enhanced lipid absorption did not increase body weight and why lipoproteins accumulate in the plasma of Ire1β-deficient mice. It is likely that plasma lipoprotein lipolysis and their clearance by other tissues are unaltered in the absence of Ire1β. This is supported by our limited studies in the liver that did not show significant differences between Ire1b−/−/Apoe−/− and Apoe−/− mice.
Hyperlipidemia is a known risk factor for atherosclerosis. Therefore, we hypothesized that sustained hyperlipidemia might enhance atherosclerosis in Apoe−/− mice. We report for the first time to our knowledge that Ire1β deficiency increases atherosclerosis in Apoe−/− mice fed a chow diet. Moreover, the disease development was accelerated when fed a western diet. Overall, there were more lesions throughout the aorta in Ire1b−/−/Apoe−/− mice compared with Apoe−/− mice fed either chow or western diets. Therefore, these studies indicate that more intestinal lipoprotein assembly and secretion can contribute to atherogenesis.
Deficiency of Ire1β also resulted in higher levels of plasma apoB48 levels. Veniant et al9 indicated that atherosclerosis is more likely related to plasma cholesterol concentration, although they showed increased postprandial atherogenicity in apoB48-only mice in the Apoe−/− background. Presence of apoB48 has been found in atherosclerotic plaques in both animal and human studies and are suspected to be atherogenic.33,34 Hence, increased plasma apoB48-containing lipoproteins in Ire1b−/−/Apoe−/− mice may render these mice more susceptible to atherosclerosis.
In summary, these studies support the hypothesis that Ire1β reduces intestinal lipid absorption. They show that Ire1β deficiency increases intestinal lipid absorption mainly because of enhanced chylomicron assembly and secretion. Further, we observed more atherosclerotic plaques in Ire1b−/−/ Apoe−/− mice. Thus, increased sustained amounts of intestinal lipoproteins in the absence of Ire1β could contribute to atherogenesis. It is likely that agents that increase expression of Ire1β might reduce hyperlipidemia and atherosclerosis.
Sources of Funding
This work was supported in part by National Institutes of Health grant DK-46900 (M.M.H.) and DK47119 (D.R.). D.R. is a Principal Research Fellow of the Wellcome Trust.
The authors thank Wei Quan for technical assistance in negative staining and electron microscopy.
In March 2012, the average time from submission to first decision for all original research papers submitted to Circulation Research was 13.2 days.
Non-standard Abbreviations and Acronyms
- apolipoprotein B
- apolipoprotein E
- fast protein liquid chromatography
- inositol requiring enzyme 1β
- microsomal triglyceride transfer protein
- Received January 5, 2012.
- Revision received April 13, 2012.
- Accepted April 26, 2012.
- © 2012 American Heart Association, Inc.
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Novelty and Significance
What Is Known?
Excessive plasma concentrations of triglycerides and cholesterol enhance the risk for several prevalent diseases, such as obesity, diabetes, and atherosclerosis.
It has been proposed that chylomicron remnants generated during the postprandial state also contribute to atherosclerosis.
What New Information Does This Article Contribute?
Inositol-requiring enzyme 1β (Ire1β) regulates intestinal lipid absorption in Apoe−/− mice.
Ire1β deficiency leads to hyperlipidemia in Apoe−/− mice.
The absence of Ire1β increases plasma concentrations of intestinally derived lipoproteins and contributed to atherogenesis.
Excess plasma lipid concentrations are risk factors for several cardiovascular and metabolic diseases. The relationship between excess lipid concentrations and cardiovascular disease is based on measurements of plasma samples collected during a fasting state. However, humans spend much of the day in a postprandial (fed) state. A confounding factor in interpreting associations between intestinally derived lipoproteins and development of atherosclerosis is the concomitant change in hepatic lipoproteins in the postprandial state. To address this issue, we took advantage of our previous observation that Ire1β is only expressed in the intestine, where it suppresses intestinal lipid absorption by regulating microsomal triglyceride transfer protein (MTP), a critical chaperone required for the assembly and the secretion of apoB-containing lipoproteins. In the present study, we show that Ire1β deficiency enhances hyperlipidemia in 1-year-old Apoe−/− mice fed a normal or saturated fat–enriched diet. We demonstrated that Ire1β deficiency increased atherosclerosis in Apoe−/− mice fed a normal laboratory diet. Moreover, atherosclerotic lesion development was accelerated during feeding of a saturated fat–enriched diet. Thus, sustained increases in intestinally derived lipoprotein concentrations attributable to the absence of Ire1β contributed to atherogenesis. It is likely that agents that increase expression of Ire1β will reduce hyperlipidemia and atherosclerosis.