Download Suppression of Hepatic XBP1s Decreases the Bile Salt Pool

Suppression of Hepatic XBP1s Decreases the Bile Salt Pool
Introduction
Cholestatic liver diseases are a prevalent group of hepatic diseases that impair
the liver from secreting bile, frequently progressing to cirrhosis and often necessitating a
liver transplantation. Two major cholestatic liver diseases, Primary Sclerosing
Cholangitis (PSC) and Primary Biliary Cirrhosis (PBC), affect over 70,000 Americans.
However, the only effective treatment for PBC is the ingestion of ursodeoxycholic acid
(UDCA), discovered three decades ago [1]. There is no effective medical therapy for
PSC. In fact, there have been no therapeutic advances in the treatment of cholestatic
liver diseases since the initiation of UDCA therapy two decades ago [2, 3]. In addition,
UDCA simply slows the progression of PBC, but does not cure the disease nor prevent
the development of cirrhosis and liver failure [4, 5]. Despite multiple drug trials
attempting to treat or cure this disease, there have been essentially no medical
advancements over the past thirty years relating to this pervasive disease.
In my study, I am looking at a new approach to develop treatments for cholestatic
liver diseases. By examining the XBP1 transcription gene pathway of the Unfolded
Protein Response (UPR) pathway, we hope to be able to develop novel treatments for
patients with cholestatic diseases. Effective treatment of cholestatic liver diseases will
also reduce the need for liver transplantations, allowing the organs to be used to treat
patients with other liver diseases. Therefore, by gathering results on mice with liver
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specific loss of XBP1, we can elucidate its effect on the bile salt pool and bile salt
toxicity.
Bile salts are produced by the liver and are secreted into bile. They are
synthesized from cholesterol in the liver and have a major function in the digestive
system: emulsifying fats to promote the digestion and absorption of the fats and fat
soluble vitamins [4, 6]. More recent research has shown, however, that these
compounds also regulate many hepatic genes and signaling factors such as glucose
and energy homeostasis [6-9].Bile salts cycle in the enterohepatic circulation, where
they are secreted from the liver via bile into the intestine, are reabsorbed in the ileum
(small intestine) and returned to the liver via the portal blood circulation [7-9]. Studying
the physiology of bile salts can help us develop treatments for a host of liver diseases
as well as lead to breakthroughs in the early detection and prevention of several other
diseases including atherosclerosis [9].
There are several types of bile salts species with varying functions, albeit the
molecular structures are relatively similar, as shown in Figure 1.
In my study using mice, the expected bile salts that will appear on the graph
would be Taurocholic Acid and Tauromuricholic Acid (Glycocholic Acid was used as a
control to measure the efficiency of my organic extraction process).
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Fig. 1. Illustrates the various bile salts of differing hydrophobicity. The bile salt
hydrophobicity results in different retention times on the HPLC analysis used in
my study.
Multiple studies have demonstrated the importance of the large number of
functions of bile salts, but the effects of bile salts on the Unfolded Protein Response
(UPR) remains essentially unknown.
The endoplasmic reticulum (ER) is pervasive in all eukaryotic cells. In the ER,
approximately one-third of all cellular proteins are modified by the addition of new
functional groups or folding, because of its metabolic capabilities in producing and
synthesizing new compounds [10]. As the ER continuously folds proteins, the amount
of incorrectly folded proteins accumulate in the lumen [11]. The stress in the ER occurs
in many prevalent diseases, including cancer, diabetes, autoimmune disorders, obesity,
liver disorders, and neurodegenerative disorders [12].
Therefore, to protect ourselves against cellular stress, humans innately have an
Unfolded Protein Response (UPR), a cellular response to ER stress that is activated
when an excess of misfolded proteins are present in a cell. The UPR is an integrated
signal transduction pathway that has three main pathways including IRE1, ATF6 p90,
and PERK [13]. The UPR has three fundamental responses: 1) to restore the normal
function of the cell by ceasing any protein translation, 2) to increase the production of
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chaperonins, regulatory proteins that provide favorable conditions for the proper folding
of other proteins and thus ensuring less protein stress will be induced upon the cell, and
3) to initiate apoptosis, cell death, if the amount of misfolded protein aggregation
becomes too excessive or prolonged.
The X-box-binding Protein 1 transcription factor (XBP1) has been previously
identified as a key regulator to the Unfolded Protein Response and is located in the
IRE1 pathway. For example, prior studies have shown that XBP1 is the mediator of
amyloid-β 1-42 peptide (Aβ) neurotoxicity, which is responsible for Alzheimer’s disease
[14].
Fig. 2. Depicts the Unfolded Protein Response pathways. In this figure, I focus on
the effects of the XBP1 transcription factor [15].
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The XBP1 transcription factor also has other functions, including governing immune
regulatory genes [16], evidenced when a knockout XBP1 mouse, lacking the protective
protein, was shown to be more susceptible to disease. Many knockout mice have
normal phenotypes until their systems are stressed. By introducing bile salts into a
murine diet, the mouse may elicit a change in phenotype and liver injury. Lee et al.
provided strong evidence that the transcription factor, XBP1, also regulates normal fatty
acid synthesis in the liver, which is a previously unknown function and represents an
unexplored area of biology [17, 18]. Regardless, all three of the Unfolded Protein
Response pathways are beneficial to the liver. Because the XBP1 transcription factor is
ubiquitous in all major organs and cells, especially heavily regulated protein-producing
cells such as the liver, a complete (-/-) knockout mouse for this gene is embryonically
lethal and the mouse dies in utero.
In order to study the effects of XBP1s in the liver, my mentor developed mice
with hepatocyte (liver cells)-specific loss of XBP1 [XBP1h (-/-) mice]. I utilized these
mice to determine the effect of XBP1 on the total amount of bile salts (bile salt pool) and
bile salt species (type of bile salts) in my mouse model.
I analyzed the physiological composition of the murine bile pool and the
distribution of the bile salts using High Pressure Liquid Chromatography (HPLC).
A recent publication demonstrated that XBP1s regulates the synthesis of fatty
acids in the liver [18]. My hypothesis is that XBP1s regulates the production of bile
salt by the liver and thus the bile salt pool. Therefore, the bile salt pool will be
reduced in XBP1h (-/-) mice.
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By showing the differing amounts and types of bile salts that appear as a
consequence of preventing XBP1 expression in hepatocytes, we are better able to
understand the hepatic metabolic pathways. This may allow us to develop drugs to treat
or cure cholestatic liver diseases and potentially other diseases that affect the liver.
Materials and Methods
Animals
Three types of mice were implemented in this experiment, a control Cre-negative floxXBP1 mouse, an experimental Cre-positive mouse, and a wild-type (normal) mouse.
The Cre-lox system is a widely used system to selectively knockout genes [19]. Both the
control and the experimental mice have flanking lox genes, which are placed on both
ends of Exon 2 on the XBP1 gene in the hepatocyte cells. With the addition of a protein
called Cre-recombinase, which is only expressed where a specific type of albumin (a
globular protein solely synthesized in the liver) presides, the flanking lox genes along
with Exon 2 are excised out. The control has the continued expression of the XBP1
gene, although the mice still contain the flox-flox genes inserted into the liver-specific
cells. Thus, Exon 2 of XBP1 is only excised in the hepatocytes, which accounts for
around 90% of the liver cells. Without the full genomic expression, the XBP1 protein
experiences a frame shift, prematurely ending the protein assemblage with the stop
codon. The Cre-lox system is illustrated in Fig. 3 below:
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Fig 3. This visual representation of the Cre-lox system depicts the basis of the
recombination in experimental XBP1h (-/-) mice.
The experimental mouse, however, is a recombinant organism obtained through
breeding. Wild-type mice were purchased and did not contain any recombinant genes.
Each mouse was housed in colony cages and was given free access to rodent chow
and water at all times. To keep the variables as constant as possible, each mouse used
is the male gender and is harvested for bile after fifteen weeks. Before the sacrifice, the
mice were not fasted and were maintained on their routine feeding schedule. All animal
protocols were approved by the university’s Animal Care and Use Committee. I was not
involved with the use of live mice. I performed experiments on mouse tissues which
were provided to me by the principal investigator or other personnel in the laboratory.
These tissues were also used for experiments by other lab personnel.
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Experimental Procedure
I used High Pressure Liquid Chromatography (HPLC) on the bile salts extracted
from mice to determine the total bile pool as well as the amount and type of each biliary
salt. We incorporated the method of Heuman to find the HPLC results [20]. This process
uses the different hydrophobicities (lipid or water solubility) of each bile salt to allow bile
salt separation based on hydrophobicity. HPLC requires the running of a buffer through
the mobile phase, which dissolves the sample of bile. This mobile phase is forced
through the HPLC hydrophobicity column, which contains the stationary phase, made of
tightly packed porous silica particles, forcing the sample to have different solubility. An
inbuilt UV lamp processes the absorbance units over time to determine the amount of
each bile salt eluted.
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Fig. 4. This graph is an example of High Pressure Liquid Chromatography (HPLC)
used for the detection and quantitation of bile salts. The AU (Absorbance Units) is
on the X-axis and retention time is on the Y-axis. Each major peak represents a
specific bile salt. The bile salts are separated by the time on the graph. The area
under the curve represents the amount of each bile salt. Total bile salts include
the sum of the area under the curve.
The buffer made for the HPLC machine requires the precise measurement of pH,
so the bile salt samples can properly ionize into compounds easily distinguished by the
UV lamp. Altering the pH even minimally significantly changes the degree of ionization
of molecules in the solution, affects their polarity, and thus changes the retention times
of each compound. Because the bile salt acidity ranges from pH 5.0 to 7.0, the buffer
must be concentrated enough to run the sample through the column, but also be diluted
enough to not harm the fragile silica based column. The resulting 5.9 liters of buffer that
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remained was sufficient for the whole eight week period, so there was no variability in
the composition of the buffer for bile salt separation.
Bile Salt Extraction
I obtained the tissues of the mice (liver, small intestine, and gallbladder). These
tissues contain approximately 95% of the bile salts in the body. To measure the
efficiency of my extractions, a 12.1mM solution of glycocholic acid, a bile salt that is not
present in mice, is added into each sample. As an internal standard, each sample
underwent many purifications to ensure that other substances present in the organs will
not corrupt the data.
HPLC Procedure
I ran the samples from the seven mice five to seven times each. By doing this, I
ensured there was sufficient data to attest to the significance of events. After injecting
each sample run into the silica column and buffer, I also added a wash solution of pure
HPLC-grade methanol was pumped through the system, cleansing the machine of any
impurities that may have been adhered to the side of the column.
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Results
Expression of Bile Salt Pool and Curves
A)
B)
*
*
*
*
C)
D)
*
*
*
*
Fig 5. Total Bile Salt Concentration, Individual Bile Salt Concentration, and
Hydrophobicity in XBP1h (-/-) mice, Control (Cre-negative) mice, and Wild-Type
(Normal) mice.
A), The total bile salt pool in the XBP1h (-/-) mice was significantly reduced compared to
the control mice (* p < 0.03) and the wild-type mice. B), The tauromuricholic acid
concentration had a trend toward being reduced, although it did not reach statistical
significance (* p = 0.1). C), The bile salt pool hydrophobicity the control mice were
significantly more hydrophobic than that from the experimental group (* p < 0.04) and
even more acutely hydrophobic in the wild-type mice. D), The experimental group’s
taurocholic concentration was also significantly less than the control group (* p < 0.004)
and even less than the wild-type group. Blue boxes represent XBP1h (-/-) mice; red
boxes represent control mice; green boxes represent wild-type mice (n=5, 7, 3 for
XBP1h(-/-), Control, and Wild-Type mice respectively).
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As shown in Figure 5a, the total bile salt pool between experimental and control
mice was significantly different, with the XBP1h (-/-) experimental mice’s bile salt pool
only containing 47% of the control mice’s bile salt pool concentration and 28% of the
wild-type mice’s bile salt pool concentration. Tauromuricholic bile salt concentrations
showed a trend toward a decrease in the experimental mice. The bile salt
hydrophobicity is significantly decreased in the experimental mice, as it was 30% more
negative than the control mice’s hydrophobicity’s value and significantly more negative
than the normal mice. The taurocholic bile salt concentration showed the greatest and
most significant decrease out of the two present bile salts. The XBP1h (-/-) mice
contained a mere 28% of the control mice’s TC value and 15% of the wild-type mice’s
TC value.
Throughout each of the sample runs, the experimental XBP1h (-/-) mice
constantly displayed significantly decreased bile salt value’s compared to the control
Cre-negative mice. The wild-type mice in this study had the highest bile salt values out
of all the samples.
Qualitative Data Regarding Tauromuricholic Acid
As the experiment progressed, I observed something interesting. The bile salt
Tauromuricholic Acid (TMC) is composed of two similar isoforms, alpha-tauromuricholic
acid (TMC α) and beta-tauromicholic acid (TMC β). There are very little functional
differences known for these highly hydrophilic bile salts, as the two slightly altered
molecules are as effective as the other in preventing taurochenodeoxycholate-induced
liver damage in rats [21]. However, it is worth noting that in a qualitative experiment,
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there was a decrease in TMC β in the control mice. Typical results are shown in the
HPLC graphs of Fig. 6:
A)
B)
Fig. 6. XBP1h (-/-) mice have increased TMC β HPLC was performed on A) XBP1h (/-) samples and B) control Cre-negative mice samples. The two graphs represent the
two TMC α and TMC β peaks. There is a significantly large increase in TMC β values in
experimental mice.
As illustrated in Fig. 6 there is a significant change that was present across all three
control mice and all five XBP1h (-/-) mice.
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Discussion
In this study, I demonstrated two main findings: (1) the loss of XBP1s in the liver
reduced the bile salt pool; and (2) changed the bile salt species. These findings are
consistent with my original hypothesis. This area of research on the XBP1 transcription
factor and its effect on the bile salt pool concentration had been previously unexplored.
We show that the XBP1h (-/-) mice displayed a significant decrease in the hydrophobic
bile salts TC and TDC, contributing to an overall decrease in hydrophobicity. This has
several important implications in normal health and cholestatic liver diseases. The
hydrophilic bile salts are more beneficial to prevent liver injury, as a hydrophobic bile
salt buildup instigates toxicity within the enterohepatic circulation. Previous studies have
shown that the hydrophilic bile salts in the liver are beneficial for the treatment of
cholestasis [22]. The hydrophilic-hydrophobic balance of the enterohepatic system is a
delicate equilibrium. Hydrophilic bile salts have been known to inhibit cholesterol
absorption, preventing the aggregation of bile salt synthesis because bile salts are
formed form cholesterol components [23]. For example, ursodeoxycholic acid (UDCA) is
a hydrophilic bile salt that has been the main source of treatment for cases of Primary
Sclerosing Cholangitis (PSC) and Primary Biliary Cirrhosis (PBC). UDCA is the only
FDA approved drug to treat these cholestatic diseases; however, it is a highly
controversial form of treatment that has been met with differing results. In Gong et al.,
there was no discernible benefit of UDCA on mortality or liver transplantations [24].
Taken together, my results suggest that the XBP1 transcription factor alters bile salt
production and thereby contributes to lower bile salt toxicity in the experimental mice.
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The control Cre-negative mice’s bile salt values increased significantly compared
with the experimental XBP1h (-/-) mice. The wild-type mice, however, consistently had
the highest bile salt values throughout all the mice. Therefore, the control mice
represented a medium between the experimental XBP1h (-/-) mice and the wild-type
mice. The flox-flox genes placed around the ends of Exon 2 of XBP1 may alter the
functionality of CYP7A1, the gene for the rate-limiting step of cholesterol catabolism for
bile salt synthesis, in some minor way, and therefore are the best control mice for Cre+
flox mice [8, 9, 10, 25, 26].
I found that, after knocking out XBP1 throughout hepatocytes, there was a
distinct effect on the bile salt pool. It is unlikely that repressing XBP1 in the liver would
increase the excretion of bile salt the stool, as the cells in question where hepatocytespecific. There are two pathways to synthesize bile salts, through the rate-limiting
enzyme Cholesterol 7 alpha-hydroxylase, regulated by CYP7A1, and through CYP27A1
[27, 8, 10]. The major bile salt secreter is encoded by ABCB11 and is the predominant
canalicular transport protein for biliary bile salt secretion controlled by these two
pathways [28]. I hypothesize that either one or both of these metabolic pathways are
regulated by XBP1 spliced, leading to a deficiency of bile salts circulating within the
enterohepatic system.
In my study, it was found that although the XBP1h (-/-) had a decreased
concentration of TMC α, it had an elevation in the levels of TMC β. The physiological
consequences between the two different types of TMC are currently unknown, but the
difference is evident and worth noting.
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Changing the bile salt pool has many broad repercussions. Bile salts are
essential for everyday bodily functions. In the liver, they are important metabolic
regulators that account for biliary secretion of lipids and intestinal nutrient absorption as
well as signaling factors responsible for maintaining homeostasis within the organism
[29]. The enterohepatic system relies on bile salts to modulate cellular signaling
pathways, such as calcium mobilization, protein kinase C activation, and cyclic AMP
synthesis [30]. In addition, a toxic concentration of bile salts is responsible for a number
of afflictions including cholestatic liver diseases, fatty liver diseases, cardiovascular
diseases, and diabetes [29, 31, 32].
Although the mice used in the study have differing species-specific bile salts
compared to humans, the findings in this study can be used to develop potential
treatments for human diseases. Mice have tauro-conjugates in their bile salts while
humans only contain glycol-conjugates within their bile salt pool. The data acquired from
this experiment can still be used, as many of the bile salt’s functions remain conserved
throughout different species.
The results in the present study advance our understanding of the XBP1
transcription factor’s functionality in bile salt synthesis pathway. Hetz et al. recently
reported that endoplasmic reticulum stress, which initiates the UPR pathway, plays a
large part in many liver diseases, many of which are caused by the buildup of bile salt
toxicity [13].
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Conclusion and Future Work
The research in the present study shows that XBP1 is intimately involved in bile
salt physiology. Primary Sclerosing Cholangitis and Primary Biliary Cirrhosis are
diseases marked by the inflammation of the bile ducts connecting to the liver, resulting
in a toxic buildup of bile salts within the liver [8, 32]. The harmful buildup can slowly
damage the surrounding tissues over time, prompting a number of detrimental states
and even lethality. However, past research had not addressed the effects of the recently
discovered stress-reducing signaling network on the liver. Therefore, we hypothesized
that XBP1, a transcription factor that regulates important aspects of the Unfolded
Protein Response as well as other metabolic pathways, plays a large part in the upkeep
of bile salt concentration. Elucidating the pathways that regulate the bile salt secretions
could provide insight into not only the inner workings of the liver but also into developing
novel therapies for treating patients afflicted with cholangitis, cirrhosis and similar
enterohepatic diseases.
Additional studies must be performed before a complete understanding of
this phenomenon occurs. We showed that the bile salt pool significantly decreased
when XBP1h was deleted in mice; however, the specific downstream mechanism(s) that
regulate bile salt synthesis/secretion remain unknown. The bile salt pool typically
decreases such as the concentration did in the study because of two main causes: (1)
there may be a deficiency in the bile production; or (2) excess amounts of bile salts are
excreted in the stool. The mechanism responsible for these findings will be delineated in
future research. Future studies will focus on CYP7A1, the rate-limiting step in bile salt
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production, and the differences in genomic coding. These studies will help further
explore and elucidate the regulatory effects of XBP1s on bile salt concentrations.
The findings shown in the present study led us to conclude that the transcription
factor XBP1 is important in regulating the bile salt pool within the enterohepatic system.
More specifically, XBP1 considerably reduced the concentrations of the species of bile
salts, and thus the total bile salt pool. However, highly effective treatments for Primary
Sclerosing Cholangitis and Primary Biliary Cirrhosis are still lacking. Therefore, new
pharmaceutical compounds that target hepatic XBP1s may potentially be useful for the
treatment of cholestatic and other liver diseases.
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