2. Introduction
Hyperlipidemia is a major risk factor associated with atherosclerosis, obesity,
diabetes, and metabolic syndrome and ensues due to overproduction or reduced
clearance of lipoproteins. There is increasing evidence that alterations in
metabolism of triglyceride-rich lipoproteins are of importance in the
pathogenesis of atherosclerosis and its clinical consequences [1]. Further,
increased hepatic very low density lipoprotein (VLDL) synthesis is the principal
defect in subjects with hyperlipidemia and is also an important component of the
dyslipidemia of diabetes and obesity [2, 3, 4]. Plasma homeostasis of lipids is
maintained by balancing production and catabolism of apoB-lipoproteins mainly by
the liver and intestine. Molecular mechanisms involved in lipoprotein
assembly by the intestine and liver requires MTP, an essential chaperone resident
in the endoplasmic reticulum, which transfers several lipids in vitro[5, 6, 7].
The endoplasmic reticulum (ER) is the major site for lipid synthesis and
apoB-lipoproteins assembly. The ER stress, which occurs due to disruption in ER
protein folding capacity, leads to activation of an evolutionarily conserved
unfolded protein response (UPR) signaling system in order to restore ER
homeostasis. The UPR allows cells to manage ER homeostasis through an adaptive
mechanism involving IRE1, protein kinase R (PKR)-like ER kinase (PERK),
and activating transcription factor 6 (ATF6) [8, 9]. These three proteins act in
parallel to transmit information across the ER membrane to decrease protein
synthesis and induce transcription factors to enhance the synthesis of
chaperones. Increasing evidence suggests that ER stress and UPR activation can
regulate cellular processes beyond ER protein folding and can play crucial roles
in lipid metabolism [10, 11, 12]. Any disturbance in ER homeostasis can stimulate
lipogenesis [13] and inhibit hepatic VLDL secretion [14, 15], leading to
hepatosteatosis. Deletion of the hepatocyte-specific Ire1a gene causes
profound hepatosteatosis and hypolipidemia in the mice upon ER stress [11].
Furthermore, transcription factor X-box binding protein 1 (XBP-1) have been shown
to directly regulate hepatic lipogenesis and lipid metabolic pathways [12].
MTP is regulated mainly at the transcriptional level [16, 17]. Because of its
essential role in lipoprotein biosynthesis and availability of in vitro
assays to measure its activity, MTP has been a favorite target to lower plasma
lipids. Several antagonists have been identified that inhibit MTP activity and
reduce plasma lipids [18, 19]. However, they are associated with substantial side
effects. It is hypothesized that toxicities associated with MTP therapy can be
avoided by tissue-specific inhibition of the intestinal MTP and sparing hepatic
MTP. In fact, several such reagents have been identified [19]. Hence, it is
timely to explore intestine specific mechanisms that control lipid absorption.
Besides transcriptional regulation, IRE1, cleaves MTP mRNA using a
novel post-transcriptional regulatory mechanism to down regulate MTP expression
[20]. Absence of Ire1 expression predisposes mice to
hyperlipidemia and atherosclerosis when fed high cholesterol and fat diet [21].
In this study, we asked whether a ubiquitously expressed homolog of
IRE1, IRE1 known to play a critical role in
the UPR or ER stress, is also involved in the regulation of MTP and intestinal
lipid absorption. Gene deletion studies have revealed that
Ire1a embryos die between 12.5 and 13 days of gestation
[22]. Hence, Ire1a floxed mice have been generated to study tissue
specific role of IRE1 [11]. We generated intestine specific
IRE1 knockout mice to study whether intestinal IRE1 regulates
plasma lipids by modulating intestinal lipid absorption.
3. Materials and methods
3.1 Materials
Infinity Cholesterol (catalog #TR13421), Infinity Triglyceride (catalog
#TR22421), and TRIzol (catalog #15596018) reagents were purchased from
Thermo Fisher Scientific (Middletown, VA). Omniscript RT (catalog #205113) kit
was purchased from Qiagen (Germantown, MD) and qPCR core kit for SYBR
Green I (catalog #10-SN10-05) was from Eurogentec (San Diego, CA). Western diet
(catalog #TD.88137) containing 17.3%, 48.5%, 21.2%, and 0.2% by weight of
protein, carbohydrate, fat, and cholesterol, respectively was purchased from
Envigo (Indianapolis, IN). All other chemicals and solvents were obtained from
Fisher Scientific (Pittsburgh, PA) or VWR International (Bridgeport, NJ).
3.2 Animals
Ire1a mice have been previously described [11] and were crossed
with Vil-CRE transgenic mice [23] to obtain heterozygous
I-Ire1a. These mice were crossed to generate homozygous
Ire1a mice. Ire1a mice were used as wild type
controls (I-Ire1a) in the study. Mice were kept at 21–23
C on a 12-h dark/light cycle (lights on: 7:00 AM–7:00 PM). To study the
effects of Western diet, 12-weeks old male I-Ire1a and
Ire1a mice (22–25 g; N = 3 per group) were fed a Western diet
for 3 weeks. Mice were fed either chow diet (LabDiet 5001) or Western diet
(TD88137, Envigo; Indianapolis, IN) containing 23.9, 48.7, 5, 0.02% (chow diet)
17, 48.5, 21.2, and 0.2% (Western diet) by weight of protein, carbohydrate, fat,
and cholesterol, respectively. Animal care and procedures were performed in
accordance with the guidelines of and approved by the Institutional Animal Care
and Use Committee of State University of New York Downstate Medical Center.
3.3 Plasma and tissue lipid measurements
Total cholesterol and triglyceride levels in the plasma and tissues were
measured using commercially available kits from Thermo Fisher Scientific
(Middletown, VA) as described previously [24]. Plasma lipoproteins were separated
by gel filtration (flow rate of 0.2 mL/min) using a Superose 6 10/300 GL column
(GE Healthcare Life Sciences, Marlborough, MA), and 200 L fractions
were collected to determine the cholesterol and triglycerides in apoB- and non
apoB-containing lipoproteins [20].
3.4 Determination of MTP activity
Small pieces (0.1 g) of liver or proximal small intestine
(~1-cm) were homogenized in low salt buffer (1 mM Tris-HCl, pH
7.6, 1 mM EGTA, and 1 mM MgCl) and centrifuged, and supernatants were used
for protein determination and MTP assay [25].
3.5 mRNA quantification
Total RNA from tissues was isolated using TRIzol. The purity of RNA was
assessed by the A/A ratio. RNA preparations with
A/A ratios more than 1.7 were used for cDNA synthesis. The first
strand cDNA was synthesized using Omniscript RT kit. Each reaction of
quantitative PCR was carried out in a volume of 20 L, consisting of
10 L of cDNA sample (1 : 100 dilution of the first strand cDNA
sample) and 10 L of PCR master mix solution containing 1X PCR
buffer from qPCR core kit for SYBR Green I. The PCR was carried out by
incubating the reaction mixture first for 10 min at 95 C followed by 40
cycles of 15 sec incubations at 95 C and 1 min at 60 C in an
ABI 7000 SDS PCR machine (Thermo Fisher Scientific, Middletown, VA). Data were
analyzed using C method, according to the
manufacturer’s instructions, and presented as arbitrary units that were
normalized to ArpP0 mRNA.
3.6 Xbp-1 mRNA splicing
A two-step RT-PCR method was used to determine Xbp-1 mRNA splicing. The
primers (Xbp1-forward 5′-ggccgggtctgctgagt-3′ and
Xbp1-reverse 5′-tccttctgggtagacctctggga-3′) were designed to
differentiate the two forms of Xbp-1, which differ by 26 nucleotides.
The PCR products were separated by 3% agarose gels.
3.7 Statistical analysis
Data were analyzed using GraphPad Prism 5 (Windows V5, GraphPad Software). Data
are presented as mean standard deviation (S.D). Comparisons of mean
values between two groups were performed by Students’ t test. Values of
P 0.05 were considered significant.
4. Results
4.1 Deletion of intestinal IRE1 does not affect ER stress
genes in the intestine or liver of chow fed mice
To determine whether deletion of IRE1 in the intestine affects ER
stress, intestinal and liver mRNA from 12 week old
Vil-Cre-Ire1a (I-Ire1a) and
Vil-Cre-Ire1a (Ire1a) mice on chow diet
was used to quantify the expression of different ER stress genes. Deletion of
Ire1a gene resulted in a significant knockdown of ~80%
in its mRNA levels in the intestine (P 0.001). However, no change
was observed in the mRNA levels of Ire1 or other ER stress
genes (Perk, Atf6, Atf4, BiP, and
Chop) in both the intestine and liver (Fig. 1A,B). Similarly, feeding of
chow diet did not result in the splicing of intestinal or hepatic Xbp-1
mRNA, another marker of ER stress, in both
I-Ire1aand Ire1a mice (Fig. 1C,D). These data show that deletion of IRE1 in the intestine does not
result in ER stress in chow fed mice.
Fig. 1.
Expression of ER stress genes in intestine specific ablated
Ire1a mice fed chow diet. 12-week old male (n = 3)
Vil-Cre Ire1a(I-Ire1a) and
Vil-Cre Ire1a (Ire1a) mice on chow diet
were fasted for 6 h and sacrificed. mRNA from intestine and liver were used to
determine expression of various ER stress genes (A and B) and Xbp-1
splicing (C and D), respectively. Values (mean SD). P 0.001 are significantly different from I-Ire1a mice. Ctr,
positive control for Xbp-1 splicing.
4.2 Plasma lipids are reduced in Ire1a mice fed chow diet
To test whether intestinal IRE1 plays a role in lipid metabolism,
12 week old I-Ire1a and
Ire1a mice on chow diet were fasted for 6 h and plasma lipids
were measured. Compared with I-Ire1a control mice, both plasma
cholesterol (Fig. 2A) and triglycerides (Fig. 2B) were significantly reduced by
~29–43% in Ire1a mice (P 0.01 &
P 0.001, respectively). The decrease in cholesterol was in both
apoB- and non apoB-containing lipoproteins (Fig. 2C) whereas decrease in
triglycerides was in mainly in apoB-containing low-density lipoprotein (LDL)/VLDL
lipoproteins (Fig. 2D). These results suggest that intestine specific deletion of
Ire1a decreases plasma lipids.
Fig. 2.
Effect of intestine specific deletion of Ire1a on
plasma lipids in chow fed mice. 12-week old male (n = 3)
I-Ire1a and Ire1a mice on
chow diet were fasted for 6 h and blood was collected through retro-orbital
bleeding. Plasma was used to measure total cholesterol (A) and triglycerides (B).
Pooled plasma from each group was separated by FPLC and lipids were measured in
the fractions (C and D). Values (mean SD) are representative from 2
independent experiments. P 0.01 and P
0.001 are significantly different from I-Ire1a mice.
4.3 Intestinal cholesterol and hepatic triglycerides are decreased
in Ire1a mice fed chow diet with no change in MTP expression
We have shown previously that deletion of IRE1 results in an increase
in the expression of intestinal MTP [20]. On the other hand, Wang et al.
[26] have shown that deletion of IRE1 in the liver results in decreased
MTP activity. To determine whether decrease in plasma lipids in
Ire1a mice on chow diet was due to any
changes in the expression of MTP, we measured MTP activity (Fig. 3A,D) and mRNA
levels (Fig. 4A,B) in the intestine and the livers of these mice. We did not
observe any significant difference in the intestinal and hepatic MTP activity and
mRNA levels between the wild type and intestine specific Ire1a gene
knockout mice. Next, we looked at the levels of lipids in these tissues that
might contribute to any changes in plasma lipids. There was a significant
decrease of 35% (P 0.5) in the intestinal cholesterol levels (Fig. 3B) in Ire1a mice compared to
I-Ire1a mice. This decrease might be due to
less uptake of dietary cholesterol from the intestinal lumen since
Ire1a mice exhibited reduced expression of
~67% in Npc1l1 levels in the intestine (Fig. 4A).
Interestingly, we also saw a significant decrease of ~40%
(P 0.05) in hepatic triglycerides (Fig. 3F) in these mice which
might be secondary to decreased absorption by the intestine. On the other hand,
no difference was seen in the intestinal triglycerides (Fig. 3C) or hepatic
cholesterol (Fig. 3E) between I-Ire1aand
Ire1a mice.
Fig. 3.
Effect of intestine specific deletion of Ire1a on MTP
activity and tissue lipids in chow fed mice. 12-week old male (n = 3)
I-Ire1a and Ire1a mice on chow diet were
fasted for 6 h and sacrificed. Intestine and liver were used to measure MTP
activity (A and D), total cholesterol (B and E), and triglycerides (C and F),
respectively. Values (mean SD) are representative from 2 independent
experiments. P 0.05 and P 0.01 are
significantly different from I-Ire1a mice.
Fig. 4.
Expression of lipid metabolism genes in intestine specific IRE1a
knockout mice fed chow diet. 12-week old male (n = 3)
I-Ire1a and Ire1a mice on
chow diet were fasted for 6 h and sacrificed. mRNA from intestine (A) and liver
(B) were used to determine expression of various lipid metabolism genes. Values
(mean SD). P 0.05 and P 0.01 are
significantly different from I-Ire1a mice.
4.4 Expression of lipid metabolism genes in the intestine and liver
of chow fed Ire1a mice
To determine whether deletion of IRE1 in the intestine affects lipid
metabolism genes in the intestine and liver, we isolated mRNA from
I-Ire1a and Ire1a mice and performed
quantitative PCR of various genes. Besides Npc1l1, deletion of
intestinal IRE1 also decreased intestinal Abca1 levels (Fig. 4A) by 46% (P 0.05) which may be due to less dietary cholesterol
being taken up by the enterocytes. We also observed decreased expression of
~49–56% (P 0.05) in
Cpt1, Ppar,
Acc1, Scd1 and Mgat2 mRNA levels in the
intestine of Ire1a mice (Fig. 4A) suggesting
that both oxidation as well as synthesis of lipids may be reduced in
Ire1a mice. Furthermore, these mice also showed a significant
reduction of 55% and 66% in Ppar and Cpt1
mRNA levels (P 0.05 & P 0.01), respectively in the
liver suggesting reduced lipid oxidation (Fig. 4B). On the other hand, levels of
Mgat2, Srebp-1c and Srebp2 were significantly
increased (P 0.01) in the livers of Ire1a mice
compared to I-Ire1a mice (Fig. 4B) suggesting that liver is
working toward increasing the synthesis of lipids to compensate for their reduced
uptake from the plasma.
4.5 Western diet affects expression of ER stress genes in intestinal
IRE1 knockout mice
Chow diet resulted in lower plasma and tissue lipids in the intestine specific
IRE1 knockout mice without any significant change in the expression of
ER stress genes or MTP (Figs. 1-4). Next, we wanted to check whether Western diet
had similar effect on the plasma and tissue lipid levels and tissue gene
expression. Feeding of Western diet for 3 weeks significantly reduced the
expression of ER stress genes in the intestine of Ire1a mice
compared to I-Ire1a mice (Fig. 5A). Expression of Atf4,
Perk, BiP and Ire1 in the intestine was
decreased by ~27–55% (P 0.01), but Atf6
expression was increased by 58% (P 0.05) in
Ire1a mice compared to I-Ire1a mice (Fig. 5A). Feeding of Western diet was associated with the splicing of Xbp-1
mRNA only in the intestine of I-Ire1amice but not in
Ire1a mice (Fig. 5C). These data show that deletion of
IRE1 in the intestine may prevent Western diet induced Xbp-1
splicing and downstream ER stress response. However, increase in the expression
of ATF6 levels in the intestine of Ire1a mice suggest that
ATF6 arm of the unfolded protein response may be activated to mitigate the ER
stress in the absence of IRE1. Next, we measured the levels of ER
stress genes in the liver to determine if deletion of intestinal IRE1
affects their expression in the liver. Expression of ER stress mRNAs
(Ire1, Perk, Atf6, Atf4,
BiP, and Chop) were increased by 36–88% (P 0.05)
in Ire1a livers (Fig. 5B). Similarly, there was an increased
splicing of Xbp-1 mRNA (Fig. 5D) in the livers of
Ire1a mice compared to
I-Ire1amice. These data suggest that Western diet has a
differential effect on the expression of ER stress genes in the intestine and
liver of Ire1a mice.
Fig. 5.
Expression of ER stress genes in intestine specific ablated
Ire1a mice fed Western diet. 12-week old male (n = 3)
I-Ire1a and Ire1a mice on
Western diet for 3 weeks were fasted for 6 h and sacrificed. mRNA from intestine
and liver were used to determine expression of various ER stress genes (A and B)
and Xbp-1 splicing (C and D), respectively. Values (mean SD).
P 0.05, P 0.01 and P
0.001 are significantly different from I-Ire1a mice. Ctr,
positive control for Xbp-1 splicing.
4.6 Plasma triglycerides are increased in Ire1a mice on
Western diet
To determine whether Western diet also affects lipid metabolism in the
intestinal IRE1 knockout mice, 12-week old I-Ire1a and
Ire1a mice on Western diet for 3 weeks were fasted for 6 h and
plasma lipids were measured. Compared with I-Ire1a mice, total
plasma cholesterol was unaffected (Fig. 6A) but plasma triglycerides (Fig. 6B)
were significantly increased by 37% (P 0.01) in
Ire1a mice. Although total plasma cholesterol was not changed,
we observed that there was an increase in the levels of cholesterol in
apoB-containing lipoproteins with a decrease in non-apoB containing lipoprotein
cholesterol (Fig. 6C). Furthermore, increase in the levels of total plasma
triglycerides was mainly due to an increase in the triglycerides in
apoB-containing lipoproteins (Fig. 6D). These results suggest that Western diet
feeding increases the levels of plasma lipids in the apoB-containing lipoproteins
in intestine specific Ire1a gene deleted mice.
Fig. 6.
Effect of intestine specific deletion of Ire1a on
plasma lipids in Western diet fed mice. 12-week old male (n = 3)
I-Ire1a and Ire1a mice on
Western diet for 3 weeks were fasted for 6 h and blood was collected through
retro-orbital bleeding. Plasma was used to measure total cholesterol (A) and
triglycerides (B). Pooled plasma from each group was separated by FPLC and lipids
were measured in the fractions (C and D). Values (mean SD).
P 0.01 are significantly different from
I-Ire1a mice.
4.7 Western diet affects tissue lipids and MTP expression in
Ire1a mice
We observed earlier that chow diet decreased the plasma and tissue lipids in
Ire1a mice without any change in MTP expression (Figs. 2-4).
Contrary to chow diet (Fig. 2C), Western diet increased the levels of plasma
triglycerides by 37% in the knockout mice compared to wild type mice (Fig. 6C).
To determine whether this increase was due to any change in MTP expression or
tissue lipids, we measured the MTP activity and mRNA as well as cholesterol and
triglycerides in the intestine and liver of
I-Ire1a and Ire1a mice.
Feeding of Western diet resulted in a significant increase of
~25% (P 0.01) in the intestinal
MTP activity (Fig. 7A) and 70% (P 0.05) in the intestinal MTP mRNA
levels (Fig. 8A) in Ire1amice compared to
I-Ire1a mice. On the other hand, there was an insignificant
decrease of 22% and a significant decrease of 56% (P 0.001) in the
levels of intestinal cholesterol and triglycerides, respectively in
Ire1a mice (Fig. 7B,C). These data suggests
that increase in plasma lipids may be due to increased secretion of
apoB-containing lipoproteins by the intestine. Next, we looked at the effect of
Western diet on the hepatic MTP expression and lipids in I-Ire1a and Ire1a mice. Contrary to increased intestinal MTP
expression, there was a reduction of around 45% (P 0.001) in the
levels of MTP activity (Fig. 7D) and mRNA (Fig. 8B) in the livers of knockout
mice. This reduction in MTP expression was accompanied by ~8-fold
accumulation in the levels of hepatic triglycerides in Ire1amice compared to I-Ire1a mice (Fig. 7F). Again, no significant
difference was seen in the hepatic cholesterol (Fig. 7E) between
I-Ire1a and Ire1a mice fed a Western diet for
3 weeks.
Fig. 7.
Effect of intestine specific deletion of Ire1a on MTP
activity and tissue lipids in Western diet fed mice. 12-week old male (n = 3)
I-Ire1a and Ire1a mice on
Western diet for 3 weeks were fasted for 6 h and sacrificed. Intestine and liver
were used to measure MTP activity (A and D), total cholesterol (B and E), and
triglycerides (C and F), respectively. Values (mean SD). P 0.01 and P 0.001 are significantly different from
I-Ire1a mice.
4.8 Expression of lipid metabolism genes in the intestine and liver
of Ire1a mice fed Western diet
Overall feeding of Western diet did not change the expression of triglyceride
synthesis genes in the intestine of I-Ire1a and
Ire1a mice (Fig. 8A). However, there was a reduction of 56%
and 44% in the expression of Cpt1 and Ppar (P 0.05), respectively in the knockout mice intestine suggesting a
lower fatty acid oxidation compared to wild type mice. Similar to chow fed mice,
we observed a decrease of 55% (0.05) in the expression of Npc1l1 levels
in the intestine of Ire1a mice fed a Western
diet (Fig. 8A) which may suggest that these mice take up less cholesterol by the
enterocytes. Next, we looked at the expression of lipid metabolism genes in the
liver. Interestingly, expression of lipid metabolism genes such as Fas,
Dgat1, Dgat2, Mgat2, Srebp-1c,
Cpt1, Ppar, Abcg8, etc. was
significantly increased by Western diet in the livers of Ire1a
mice (Fig. 8B). Besides, lipid metabolism genes, there was also an increase of
~3.7-fold in the expression of Fgf21 in the livers of
Ire1a mice (Fig. 8B). These results along with decreased
activity and expression of MTP may explain the increased levels of triglycerides
in the liver of Ire1a mice fed a Western diet.
Fig. 8.
Expression of lipid metabolism genes in intestine specific IRE1a
knockout mice fed Western diet. 12-week old male (n = 3)
I-Ire1a and Ire1a mice on Western diet for 3
weeks were fasted for 6 h and sacrificed. mRNA from intestine (A) and liver (B)
were used to determine expression of various lipid metabolism genes. Values (mean
SD). P 0.05, P 0.01, and P 0.001 are
significantly different from I-Ire1a mice.
5. Discussion
Intestinal and hepatic MTP expression plays an essential role in regulating
plasma lipid and lipoprotein levels. MTP expression is controlled at
transcriptional and post-transcriptional levels and we have shown that intestinal
IRE1 down regulates MTP expression to decrease plasma lipid levels [20].
In this study, we asked whether ubiquitously expressed homolog of IRE1,
IRE1 is also involved in the regulation of plasma lipid metabolism. The
molecular and physiological implications of ER stress sensor, IRE11 as
well as its mechanistic action in metabolic disorders have been well described
[27, 28]. Gene deletion studies have revealed that Ire1a embryos die
between 12.5 and 13 days of gestation [22]. Therefore, we generated intestine
specific IRE1 knockout mice to study its role in plasma lipid levels.
IRE1 knockout mice (Ire1a and Ire1a) fed with
either chow diet or Western diet for 3 weeks did not result in any significant
changes in body weight (data not shown). Analysis of gene expression in the
intestine and liver revealed that deletion of intestine specific IRE1
does not affect the expression of ER stress gene markers nor Xbp-1 splicing on
chow diet (Fig. 1). In contrast, Zhang et al. [29] reported increased expression
of Chop and other ER stress markers in colon epithelial cells of intestine
specific Ire1a mice. This discrepancy may be due to the differences in
either the age (16–18 weeks) or the sex (females) of mice used in the study. In
the current study, we used 12 weeks old male mice to study the role of
IRE1 in the regulation of plasma lipids. It was reported that females
were more susceptible to develop intestinal dysfunction such as colitis in the
absence of intestinal IRE1[29]. These authors also reported increased
mortality only after the age of around 16–18 weeks suggesting that intestinal
dysfunction and, therefore, ER stress may be exacerbated in the later stages of
life in these mice. We noticed decreased Xbp-1 splicing in the intestine of
Ire1a mice fed a Western diet for 3 weeks (Fig. 5C). This is consistent
with the reduced expression of IRE1 in the intestine. On the other
hand, Western diet increased Xbp-1 splicing in the intestine of wild type mice.
We have shown previously that IRE1 does not contribute to the increase
in Xbp-1 splicing in the intestine of Western diet fed mice [20]. During chronic
stress, Zhang et al. [29] also reported a decreased expression of spliced variant
of Xbp-1 in the intestinal epithelial cells of 16–18 weeks old female
Ire1a mice. The combined data suggests that chronic
ER stress either due to Western diet feeding or older age of mice augments Xbp-1
mRNA splicing and IRE1 cannot compensate for the absence of
IRE1 in the splicing of Xbp-1 mRNA.
In addition, we have shown previously that absence of IRE1 in the
intestine of chow diet fed mice does not change plasma lipids significantly [20].
In the present study, our data indicate that deletion of intestine-specific
IRE1 decrease plasma cholesterol and triglycerides in chow diet fed
mice and this decrease was independent of changes in intestinal MTP activity or
mRNA levels (Figs. 2-4). These mice also showed a reduction in intestinal
cholesterol levels. Determination of mRNA levels in the chow fed Ire1a
suggest a decrease in the intestinal Npc1l1 and Abca1 levels suggesting that both
uptake as well as efflux of cholesterol by the enterocytes may be reduced in the
knockout mice resulting in reduced plasma cholesterol levels. Interestingly, our
data revealed a decrease in hepatic triglyceride levels. This reduction in
hepatic triglycerides may be due to reduced absorption by the intestine.
Quantification of mRNA levels suggested changes in the expression of several
lipid metabolism genes in the liver which may be secondary to the changes in the
lipid absorption by the intestine. This increase may be due to compensatory
changes in the liver to boost up the synthesis because of less lipids coming from
the intestine. These results are consistent with the intestine-specific MTP
knockout mice which show a decrease in hepatic lipid levels with a compensatory
increase in lipogenic genes due to less lipid absorption from the intestine [30, 31].
Using liver specific IRE1 knockout mice, Wang et al. [26] showed that
IRE1 in the liver reduces plasma lipids primarily due to a defective
triglyceride-rich VLDL secretion. However, they did not find any defects in
lipogenesis or apoB synthesis and secretion in the L-Ire1a hepatocytes.
Furthermore, similar to our data, these authors reported that expression of MTP
was not affected by the deletion of IRE1. However, they noticed a
decrease of MTP activity in the liver due to reduction in the expression of
protein disulfide isomerase (PDI), a subunit of MTP that is
necessary for its normal activity [32]. It is possible that IRE1 has
different roles in regulating MTP activity in the intestine and liver to affect
lipid metabolism.
Contrary to chow diet, Western diet caused an increase in plasma and a decrease
in the intestinal triglycerides level in Ire1a mice (Figs. 6,7). This
may be due to increased MTP activity and mRNA levels in the intestine of these
mice. Interestingly, we observed increased triglycerides accumulation in the
livers of Ire1a mice. This increase was accompanied by a decreased MTP
activity and mRNA expression in the livers of Ire1a mice fed a Western
diet (Figs. 7,8). Additionally, several lipogenic genes, in conjunction with
decreased MTP expression, were up-regulated in the livers of Ire1a mice
fed a Western diet which may explain the increased accumulation of triglycerides
in these mice. Our results also show that Western diet increased the expression
of ER stress genes and Xbp-1 mRNA splicing in the livers of Ire1a mice
(Fig. 5B,D). Hepatic steatosis is known to cause increased expression of spliced
variant of Xbp-1 mRNA [33]. However, deletion of XBP-1 or IRE1 in the
liver is also known to cause hepatic steatosis [11, 34]. It is possible that
active XBP-1 (spliced variant) is essential to regulate hepatic lipids and its
absence leads to hepatic steatosis. On the other hand, increased expression of
spliced variant of Xbp-1 mRNA may be a protective mechanism to counteract the
effects of higher lipids in the liver due to steatosis. How intestine specific
IRE1 deletion causes the dramatic changes in the expression of ER
stress or lipogenic genes in the liver needs further investigation.
In summary, we identified an intestine specific role of IRE1 in
regulating plasma lipid levels. The regulatory mechanism is independent of MTP
activity or expression in chow diet mice. However, similar to IRE1,
intestinal IRE1 may be required to reduce lipid absorption and MTP
expression on a Western diet. In future studies, it will be important to
determine whether IRE1 directly regulates MTP expression similar to
IRE1 by an internal cleavage of its mRNA [20]. In addition, comparing
data from L-Ire1a and Ire1a mice suggest that IRE1
may act differentially on the MTP expression in the livers and intestines to
exert its effects on plasma lipids. Taken together, our findings provide novel
insights into regulation of lipid homeostasis by modulating intestinal
IRE1. Thus, manipulation of the intestine specific IRE1
mediated signaling may provide a unique approach to treat hyperlipidemia.
6. Author contributions
JI contributed to the conception and design of the study, conducted
experiments, performed data analysis and interpretation and wrote the manuscript.
AB and AAQ performed data analysis and interpretation and wrote the
manuscript.
7. Ethics approval and consent to participate
Animal care and procedures were performed in accordance with the guidelines of
and approved by the Institutional Animal Care and Use Committee of State
University of New York Downstate Medical Center.
8. Acknowledgment
Not applicable.
9. Funding
This work was supported in part by an American Heart Association Grant-in-Aid
(12GRNT9690010), KAIMRC grants (RA16-024-A and RA17-013-A) and the International
Collaboration Initiative grant from the Ministry of Education (RA20-005-A,
Project #230) to J.I.
10. Conflict of interest
The author declares no conflict of interest.
Abbreviations
ApoB, apolipoprotein B; ATF6, activating transcription factor 6; ER, endoplasmic
reticulum; HDL, high density lipoproteins; IRE1, inositol-requiring
transmembrane kinase/endoribonuclease 1; IRE1,
inositol-requiring transmembrane kinase/endoribonuclease 1 ; LDL, low
density lipoproteins; MTP, microsomal triglyceride transfer protein; PDI, protein
disulfide isomerase; PERK, protein kinase R (PKR)-like ER kinase; UPR, unfolded
protein response; VLDL, very low density lipoproteins; XBP-1, X-box binding
protein 1.