Differential and organ-specific functions of organic solute transporter α and β in experimental cholestasis

Background & Aims Organic solute transporter (OST) subunits OSTα and OSTβ facilitate bile acid efflux from the enterocyte into the portal circulation. Patients with deficiency of OSTα or OSTβ display considerable variation in the level of bile acid malabsorption, chronic diarrhea, and signs of cholestasis. Herein, we generated and characterized a mouse model of OSTβ deficiency. Methods Ostβ-/- mice were generated using CRISR/Cas9 and compared to wild-type and Ostα-/- mice. OSTβ was re-expressed in livers of Ostβ-/- mice using adeno-associated virus serotype 8 vectors. Cholestasis was induced in both models by bile duct ligation (BDL) or 3.5-diethoxycarbonyl-1.4-dihydrocollidine (DDC) feeding. Results Similar to Ostα-/- mice, Ostβ-/- mice exhibited elongated small intestines with blunted villi and increased crypt depth. Increased expression levels of ileal Fgf15, and decreased Asbt expression in Ostβ-/- mice indicate the accumulation of bile acids in the enterocyte. In contrast to Ostα-/- mice, induction of cholestasis in Ostβ-/- mice by BDL or DDC diet led to lower survival rates and severe body weight loss, but an improved liver phenotype. Restoration of hepatic Ostβ expression via adeno-associated virus-mediated overexpression did not rescue the phenotype of Ostβ-/- mice. Conclusions OSTβ is pivotal for bile acid transport in the ileum and its deficiency leads to an intestinal phenotype similar to Ostα-/- mice, but it exerts distinct effects on survival and the liver phenotype, independent of its expression in the liver. Our findings provide insights into the variable clinical presentation of patients with OSTα and OSTβ deficiencies. Lay summary Organic solute transporter (OST) subunits OSTα and OSTβ together facilitate the efflux of conjugated bile acids into the portal circulation. Ostα knockout mice have longer and thicker small intestines and are largely protected against experimental cholestatic liver injury. Herein, we generated and characterized Ostβ knockout mice for the first time. Ostα and Ostβ knockout mice shared a similar phenotype under normal conditions. However, in cholestasis, Ostβ knockout mice had a worsened overall phenotype which indicates a separate and specific role of OSTβ, possibly as an interacting partner of other intestinal proteins.


Lay summary
Organic solute transporter (OST) subunits OSTa and OSTb together facilitate the efflux of conjugated bile acids into the portal circulation. Osta knockout mice have longer and thicker small intestines and are largely protected against experimental cholestatic liver injury. Herein, we generated and characterized Ostb knockout mice for the first time. Osta and Ostb knockout mice shared a similar phenotype under normal conditions. However, in cholestasis, Ostb knockout mice had a worsened overall phenotype which indicates a separate and specific role of OSTb, possibly as an interacting partner of other intestinal proteins.

Introduction
Bile acids facilitate the intestinal digestion and absorption of fats and fat-soluble vitamins. Bile acids are synthesized in hepatocytes from cholesterol via several enzymatic steps that form the primary bile acids cholic acid and chenodeoxycholic acid. The first and rate-limiting step of this cascade is mediated by CYP7A1. Bile acids are subsequently conjugated with amino acids glycine and taurine to form glycocholic acid, taurocholic acid, glycochenodeoxycholic acid and taurochenodeoxycholic acid. 1 A portion of primary bile acids are converted into the secondary bile acids deoxycholic acid, lithocholic acid and ursodeoxycholic acid by gut bacteria in the intestine. 1 Compared to humans, mice have a more hydrophilic bile acid composition as they can also synthesize (a-, bor U-) muricholic acid from chenodeoxycholic acid.
Tight regulation of bile acid homeostasis prevents intracellular accumulation of toxic bile acids, which can disrupt membranes, and lead to generation of reactive oxygen species and initiation of apoptosis. 2 The nuclear farnesoid X receptor (FXR) plays a central role in regulating several genes involved in the enterohepatic circulation of bile acids. Intestinal FXR increases gene expression of fibroblast growth factor (FGF) 19, the human homolog of mouse FGF15, upon binding by bile acids. 3  rate-limiting enzyme in bile acid synthesis, CYP7A. 3 In addition, activation of FXR also protects against bile acid overload in both enterocytes and hepatocytes. This is achieved by inhibiting bile acid influx via downregulation of the apical sodium-dependent bile acid transporter (ASBT) and the hepatic uptake transporter sodium taurocholate cotransporting polypeptide and stimulating export of bile acids by upregulation of efflux transporters, such as the bile salt export pump and the organic solute transporter ab (OSTa-OSTb). 4 OSTa-OSTb transports conjugated bile acids across the basolateral membrane of enterocytes into the portal circulation. [5][6][7] This transporter is a heterodimer that consists of 2 distinct subunits; a and b, 8 encoded by 2 different genes, SLC51A and SLC51B, located on separate chromosomes. The a-subunit consists of 340 amino acids with 7 transmembrane domains, while the beta-subunit only has 128 amino acids and includes 1 transmembrane domain. 6 Heterodimerization of the 2 subunits leads to increased stability of the proteins and is necessary for plasma membrane trafficking and transport activity. 9 OSTa-OSTb functions in cellular efflux of both conjugated bile acids and steroid hormones, independently of the sodium gradient. 7 Moreover, in vitro studies show that OSTa-OSTb is able to mediate both cellular efflux and influx, dependent on the concentration gradient of the substrate and extracellular pH. 7 Highest expression levels of OSTa-OSTb are detected in the distal part of the ileum. However, OSTa-OSTb also shows expression in other tissues involved in bile acid homeostasis, such as the kidney and liver, and tissues involved in steroid hormone homeostasis. 7 Of note, OSTa and OSTb are expressed with highly varying protein ratios and their transcriptional regulation is poorly correlated. 7 The relevance of this is not yet known.
To elucidate the physiological role and pathophysiological implications of OSTa deficiency, Osta -/mice have previously been generated. [10][11][12][13][14] Knockout of the Osta gene leads to complete loss of the OSTa protein, strongly reduced OSTb, 10,11,13 and results in impaired intestinal bile acid absorption and bile acid accumulation in enterocytes. 11 Compared to control mice, Osta -/mice display an ameliorated liver phenotype upon bile duct ligation (BDL), and this has been attributed to increased urinary bile acid excretion. 14 Bile acid accumulation and associated histological changes in the intestine are prevented in Osta -/mice that also lack Asbt while Fxr depletion did not resolve the phenotype of Osta -/mice. While mutations in the Asbt gene are known to cause bile acid malabsorption in humans, 15 genetic defects in Asbt do not account for all hereditary cases of bile acid malabsorption. 16 In 2019, 2 brothers were identified with a frameshift mutation in the OSTb/SLC51B gene causing impaired bile acid transport activity. 17 These patients had diarrhea, fat-soluble vitamin deficiencies and features of cholestasis, including moderately increased levels of the liver enzymes alanine aminotransferase (ALT), aspartate aminotransferase (AST) and gamma-glutamyltransferase (GGT). 17 Due to the limited availability of biospecimens from these 2 patients, little is known about the consequence of OSTb deficiency in humans. Recently, the first OSTa-deficient patient was identified; this patient had diarrhea and cholestasis, 18 which is not observed in Osta -/mice. 11,14 The OSTa-OSTb complex has an overall topology similar to the heteromeric structure of G-protein coupled receptors associated to receptor activity-modifying proteins 9,19 where OSTa adopts a 7-pass transmembrane structure, and OSTb is a transmembrane protein that crosses the membrane once. OSTb expression is necessary for glycosylation and trafficking of OSTa to the plasma membrane as well as for functional bile acid transport, 5,9,20 but whether its function is restricted to this chaperone function is unknown. Therefore, an OSTb knockout mouse model was generated to study the role of OSTb and to analyze whether deficiency of Ostb in mice affects cholestatic liver injury.

Materials and methods
For further details regarding the materials and methods used, please refer to the CTAT table and supplementary information.

Animals
Ostb -/mice were generated in C57BL/6J mice by precise targeted deletion via CRISPR/Cas9, which resulted in a large deletion in exon 3 of the Ostb (Slc51b) gene. To this end, 2 single-guide RNA (sgRNA) target sequences in the Ostb gene were selected and inserted in a pDR274 gRNA cas9-guide plasmid. The sgRNA were synthesized in vitro, purified and microinjected together with Cas9 mRNA into 1-cell stage wild-type embryos. These mice were backcrossed once to wild-type mice and resulting Ostb +/animals were crossed to create Ostb -/and wild-type littermates for analysis. Sequencing was performed to confirm the exact genotypes of the mutated Ostb gene and to analyze whether mutations occurred in potential off-target genes, which was not the case. Osta -/mice were generated by Rao et al. 13 and purchased from the Jackson Laboratory. Male and female Osta -/-, Ostb -/and control wild-type C57BL/6J mice (Janvier Labs) were housed under a 12 h light/dark cycle and bred in the Animal Research Institute Amsterdam. Mice were fed with normal chow diet and given ad libitum access to water. The study design, animal care and handling were approved by the Institutional Animal Care and Use Committee of the University of Amsterdam (Amsterdam, The Netherlands).

Cholestatic mice models
Wild-type and Ostb -/female and male adult mice (littermates) 8-12 weeks of age were subjected to a common BDL as previously described. 21 All surviving mice (both males and females) were sacrificed at day 5 because of animal welfare regulations (body weight loss >15%). A second cohort of male mice, including wild-type, Osta -/and Ostb -/adult (age 20-30 weeks) mice, were sacrificed 2 days after BDL. In a third cohort of mice, cholestasis was induced by supplementing the chow diet (D12450B1, Open Source Diets, USA) with 0.1% 3,5-diethoxycarbonyl-1,4dihydrocollidine (DDC, Sigma) during 8 days. 22 In indicated experiments, DDC diet was initiated 2 weeks after administration via the tail vein of 2x10 12 adeno-associated virus serotype 8 (AAV8) particles/kg encoding codon optimized mouse OSTb (Vectorbuilder). All mice were sacrificed under anesthesia and blood, bile and tissues were collected as described in the supplementary information.

Statistical analysis
Data are provided as mean ±SD with individual points shown in dots. Differences between groups were analyzed using a one-way ANOVA test, and Dunnett's test to compare with the wild-type littermates or Sidak's multiple comparisons test. Differences in survival were assessed using a log-rank test. Statistical significance was considered at p <0.05(*). Graphs were generated using GraphPad Prism software (version 8.0.2; GraphPad Software Inc.).
Differences in microbiota a diversity were tested using ANOVA.
Permanova was used to test compositional differences in terms of Bray-Curtis dissimilarity and Weighted Unifrac distances. Differential abundance of taxa was tested using DESeq2. 23

Generation of OSTb knockout mice
To study the role of OSTb in mice, targeted deletion was performed using CRISPR-Cas9, resulting in a 190 base pair deletion in exon 3 of the Ostb gene (Fig. 1A). Osta and Ostb mRNA were not expressed in Osta -/and Ostb -/mice, respectively (Fig. 1B). Western blotting confirmed the complete absence of OSTa and OSTb protein in Osta -/and Ostb -/mice, respectively (Fig. 1C). In line with previous Osta -/studies, we found that Osta -/mice lack the OSTa protein and have strongly reduced OSTb protein expression, 10,11,13 while Ostb -/mice lack both the OSTb protein as well as the OSTa protein. Consistent with the western blot, immunohistochemistry showed protein expression of OSTb on the basolateral membrane of ileal enterocytes in wild-type mice, while this signal was absent in Ostb -/mice (Fig. 1D).
Phenotype of Osta -/and Ostb -/mice Both Osta -/and Ostb -/mice are viable and showed no obvious change in appearance and growth. Crossing heterozygous Ostb +/mice produced a Mendelian distribution of wild-type and knockout genotypes. In contrast to the OSTb-deficient patients, Ostb -/mice showed no signs of diarrhea. Only a trend towards a modestly increased plasma level of the liver enzymes ALT (p = 0.073) and alkaline phosphatase (ALP; p = 0.075) was detected and AST levels were unchanged (Fig. 2a). Osta -/mice showed no significant change in body weight at 4 and 8 weeks after birth in both females and males. Likewise, Ostb -/mice did not demonstrate altered body weight except for 8-week-old females that showed a modest reduction in body weight compared to wildtype littermates (Fig. 2B). The length and weight of the small intestine were significantly and similarly increased in the Osta -/and Ostb -/mice in both 4-and 8-week-old mice (Fig. 2C,D). The weight per length had a tendency to increase in the Osta -/and Ostb -/mice that were 4 weeks of age, and was significantly increased in 8-week-old male mice and female Osta -/mice (Fig. S1A). Liver weight and kidney weight were not changed in the Osta -/and Ostb -/mice ( Fig. S1B,C). The length, weight and weight per length of the colon were not altered in Osta -/and Ostb -/mice ( Fig. S1D-F). The small intestine phenotype was preserved in older Ostb -/mice (32-37 weeks) (Fig. S1G).
Altered ileal histology in Osta -/and Ostb -/mice Analysis of the ileum showed an altered histology in Osta -/and Ostb -/male and female mice (Fig. 3A, Fig. S2A). While the ileum of wild-type mice comprises normal-appearing long, thin villi, Osta -/and Ostb -/mice exhibit blunted villi and elongated crypt depth. This altered ileal histology is similar between Osta -/and Ostb -/mice. Quantification of villus height showed a 25% reduction in 4-week-old female and a (not significant) 21% reduction in male Ostb -/mice compared to wild-type littermates (female mice: Ostb -/-121.7 lm ± 24.45 vs. wild-type 163.4 lm ± 25.20) (male mice: Ostb -/-115.5 lm ± 17.66 vs. wild-type 146.8  lm ± 12.20) (Fig. 3B). Additionally, crypt depth was significantly increased at both 4 weeks of age by 57% and 38% in female and male Ostb -/mice respectively, and 8 weeks of age by 83% and 64% in female and male Ostb -/mice respectively (Fig. 3C). As a result of the increased crypt depth and decreased villus height, the ratio was significantly decreased in Osta -/and Ostb -/mice (Fig. S2B). The top of the ileal villi of Osta -/and Ostb -/mice have increased numbers of mucus-filled vacuoles (Fig. 3D). Furthermore, intestinal proliferation was determined using phosphohistone H3 staining and demonstrated a more widespread distribution along the villi in both Osta -/and Ostb -/mice compared to wild-type mice probably due to the increased crypt depth (Fig. 3E). Other parts of the small intestine, the duodenum and jejunum, were not histologically altered.
Altered expression of differentiation markers in enterocytes of Osta -/and Ostb -/mice Caudal type homeobox 2 (CDX2) induces transcription of several genes implicated in intestinal differentiation and epithelial cell maturation. [24][25][26] Ostb -/mice showed a significant decrease in Data are expressed as the mean ±SD with individual points shown in dots (n = 7-13 mice per group). Statistical analysis was done using a one-way ANOVA test and Dunnett's test to compare with wild-type littermates. *Indicates p values of <0.05. ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BW, body weight; Osta, organic solute transporter alpha; Ostb, organic solute transporter beta; wk, week. expression of Cdx2 in the ileum in males at 4 and 8 weeks of age (41% and 50% reduction, respectively), and a similar decreased trend is seen in 4-week-old Ostb -/females (−31%; p = 0.15) (Fig. 4A, Fig. S3A). Osta -/and Ostb -/mice showed no change in mRNA expression of Muc2, a marker for goblet cells, and Lysozyme, a marker for Paneth cells (Fig. S3E,F). However, 4-week-old female and male Ostb -/mice had a significantly decreased expression of sucrase-isomaltase (Sis; 65% and 63% reduction,  respectively) (Fig. 4B, Fig. S3B) which was confirmed by determining SIS protein levels by immunostaining (Fig. S3C). The change in Sis expression was still observed in 8-week-old male mice (70% reduction; p = 0.0019 and 0.0013), but not in female mice (−25%; p = 0.62 and -34%; p = 0.74 for Osta -/and Ostb -/mice, respectively). Similarly, mRNA expression of Arginase2 had a tendency to decrease in Ostb -/mice at both 4 and 8 weeks of age (Fig. S3D). Neonatal markers Ass1 and Lct were not changed at mRNA levels (Fig. S3G,H). Similar findings were obtained in Osta -/mice. Together this indicates that deficiency of OSTb but also OSTa mainly affects epithelial cells in villi leading to incomplete differentiation.
Bile acid concentration and composition in circulation and excretory systems Next, we investigated the effect of Ostb deficiency on concentrations and composition in the circulation and excretory  systems. Osta -/and Ostb -/mice showed unaltered bile acid concentration and composition in bile or plasma. Furthermore, no increased bile acid excretion in urine or feces was observed and the bile acid hydrophobicity index of bile was unchanged (Fig. S6).  showed significant differences in bacterial composition between groups as shown in the principal coordinates analysis plots ((PERMANOVA p = 0.001, R2 = 0.19; Fig. S7B). Weighted Unifrac analysis, which takes the relatedness of the microbes into account, did not show significant differences, indicating the microbiota are more similar at higher taxonomic ranks. Comparing abundances of taxa in Ostb -/mice with both wildtype mice and Osta -/mice shows a decrease of Lactobacillus, various Lachnospiraceae and Candidatus_Saccharimonas and increase of Bifidobacteria and Faecalibaculum (Fig. S7C).
Ostb -/mice show lower survival rates while displaying hepatoprotective effects during BDL-induced liver injury While OSTb-and OSTa-deficient patients show features of cholestatic liver injury, 17,18 OSTa-deficient mice display attenuated liver disease upon induction of cholestasis by ligation of the common bile duct. 14 Therefore, we wondered whether challenging Ostb -/mice by inducing cholestasis would affect liver injury. To this end, we performed experiments with 2 distinct cholestasis models; common BDL and a 0.1% DDC-containing diet. Both models revealed an unexpected phenotype specifically in Ostb -/mice. First, 18 Ostb -/mice (8 male) and 20 wild-type littermates (10 male; 8-10 weeks of age) were subjected to common BDL (Fig. 5A). While all wild-type mice survived, 40% of the Ostb -/mice died within 5 days ( Fig. 5B; p = 0.02 log-rank test). No difference in body weight loss was observed in surviving Ostb -/mice compared to wild-type littermates (Fig. S8A). Remarkably, livers of surviving Ostb -/mice were completely devoid of necrotic areas, which covered 15-20% of the area in wild-type mice ( Fig. 5C and Fig. S8E). A clear reduction was observed in bile acid levels (62%), plasma bilirubin (42%) and in cholesterol (38%) levels in the surviving Ostb -/mice, while plasma ALT, ALP and AST levels were unchanged (Fig. 5D,E and Fig. S9A,B). Taurobetamuricholic acid levels are increased in Ostb -/mice compared to wild-type littermates (Fig. 5F) proliferation (Afp [p = 0.14]), but not Cyp7a1 and IL6 tended towards being reduced in surviving Ostb -/mice compared to wild-type mice after BDL (Fig. 5G, Fig. S8D). The high mortality upon BDL in Ostb -/mice was confirmed in a second experiment with a group of 3 Ostb -/mice (male, 20-30 weeks old). In this experiment we also included wild-type littermates and Osta -/mice (Fig. 5H). On day 3, animals were sacrificed due to severe symptoms of distress, including hunched posture and lethargy, specifically presented by  the Ostb -/mice. Furthermore, the contents of the stomach and the intestines were dark colored and were located throughout the small intestine while the small intestine of both Osta -/mice and wild-type mice showed a normal color and contained less alimentary matter ( Fig. 5I and Fig. S8F). Remarkably, the cages of Ostb -/mice contained considerably less feces compared to the cages of Osta -/mice (data not shown). In line with results of the first BDL experiment, examination of the liver suggested a protective effect in both Osta -/and Ostb -/mice with respect to liver damage due to BDL, with obvious pre-necrotic areas in the wildtype animals (Fig. 5J).
Ostb -/mice show lower body weight gain while displaying hepatoprotective effects when challenged with a DDC diet After 8 days on a DDC diet, Ostb -/mice showed marked body weight loss compared to wild-type and Osta -/mice (Fig. 6A). In contrast, Mcp-1 levels were significantly lower in Ostb -/mice when compared with wild-type littermates and a significant reduction was found in AST in Osta -/and Ostb -/mice when compared with wild-type mice (Fig. S9A,B) while plasma bilirubin, ALP and ALT levels as well as a-Sma, Col1a1 and Afp expression remained unchanged (Fig. S9B). Intestinal Asbt expression was decreased and Fgf15 expression increased in Osta -/and Ostb -/mice also under these cholestatic conditions (Fig. 6B). The discrepancy between overall health status and (selected) markers for liver damage mimics the BDL phenotype and suggests that, in Ostb -/mice, an extrahepatic phenotype is unmasked under cholestatic conditions which is distinct from Osta -/mice. The increased weight loss in Ostb -/mice was confirmed in a second DDC-induced cholestasis experiment where we tested the role of hepatic OSTb (Fig. 6C). To this end, we included a group that received AAV8 encoding mouse Ostb 2 weeks prior to the onset of the diet. A second difference with the first DDC experiment was that we briefly switched to control chow on day 5-6 and 9-10 to allow recovery of body weight and continued with DDC diet afterwards for another 2.5 days. Body weight loss was more severe in Ostb -/mice than wild-type mice (Fig. 6C). Body weight differences across the entire experiment are calculated as area under the curve (%.day) and were -86.07 ± 5.25 in mice expressing endogenous OSTb and -129.6 ± 10.9 and -127.0 ± 9.34 in DDC-fed Ostb -/mice (respectively mock injected or treated with OSTb-AAV8) (Fig. 6C). Hepatic expression of OSTb was confirmed in the latter group (Fig. 6D). Also, cholestatic Ostb -/mice displayed elongated small intestines (irrespective of restored hepatic OSTb expression) (Fig. 6E), while no difference in liver weight was present (Fig. 6F). This indicates that the increased body weight loss seen in cholestatic Ostb -/mice likely has an extrahepatic origin.

Discussion
Here, we generated OSTb-deficient mice and show that disruption of OSTb results in profound ileal morphological changes. When unchallenged, no major differences are observed between Osta -/and Ostb -/mice and Ostb -/mice are phenocopying Osta -/mice. Our results are mostly in line with previous Osta -/studies, 10,11,13,14,20,27,28 suggesting that OSTa and OSTb function in the same manner in bile acid homeostasis under normal conditions. However, under cholestatic conditions, Ostb -/mice have a worsened phenotype, a significant lower survival rate and lower body weight compared to both wild-type and Osta -/mice. This phenotype is independent of hepatic OSTb expression status. As the contents of the intestine and stomach of Ostb -/mice were dark colored, while Osta -/mice were indistinguishable from wild-type littermates, an intestinal origin of this phenotype is likely. Furthermore, these data indicate that there might be a difference between the function of OSTa and OSTb. The OSTb-deficiency phenotype under cholestatic conditions does not relate to liver damage since the lower survival rates of Ostb -/mice do not seem to correlate with histology and markers of bile acid-induced liver injury. The Ostb knockout mice even showed some level of protection against liver injury during cholestasis by BDL, similar to Osta -/mice. The discrepancy in content in the colon vs. the stomach of cholestatic Ostb -/mice may point to an intestinal motility phenotype. Several papers suggest a link between cholestasis and/or altered bile acid signaling and alterations in intestinal transit via 3 possible mechanisms. [29][30][31][32] First, the endogenous opioid system has been demonstrated to be activated in cholestatic conditions in mice, leading to decreased intestinal transit. 29 Therefore, induction of cholestasis may reveal or enhance an intestinal mobility phenotype in Ostb -/mice. Second, activation of TGR5, the GPCR for bile acids, is essential for peristalsis and gastric emptying, possibly via induction of glucagon-like peptide-1 secretion. 30,31 Ostb -/mice may have reduced TGR5 activation as the bile acid pool is likely reduced due to chronically elevated Fgf15 expression. Third, NGM282, an FGF15/19 mimetic has prokinetic activity itself. 32 Chronic overexpression of FGF15, as seen in Osta -/and Ostb -/mice may lead to desensitization of FGFR/KLB, just as chronic FGF23 overexpression desensitizes this receptor complex. 33 A rapid reduction in FGF15, as would occur during cholestasis may then lower intestinal motility to pathologically relevant levels. Although such mechanisms could contribute to the intestinal phenotype of Ostb -/mice in cholestatic conditions, it remains unclear why this phenotype is not exposed in Osta -/mice, which are largely indistinguishable with regard to bile acid homeostasis. This suggests that OSTb might have another function besides forming a bile acid efflux transporter upon heterodimerization with OSTa. Early after the cloning of OSTa-OSTb it was postulated that OSTb may function as a chaperone or regulatory subunit for other proteins 6 as the topology of OSTa-OSTb is similar to that of G-protein coupled receptors associated to receptor activitymodifying proteins. 34 This may also explain why the regulation of gene expression of these 2 subunits is so different. 7 For example, hepatic upregulation of OSTb expression is much higher than that of OSTa in patients with primary biliary cholangitis 35 and in obstructive cholestasis. 36 Finally, the modest but evident differences in microbial composition may lead to or reflect differences in intestinal function. Ostb -/mice were more sensitive to the DDC diet than wild-type or Osta -/mice. Ostb -/mice lost significantly more body weight which may be related to the altered microbiota as this could lower the efficiency of energy harvest. 37 This would also explain why the effect is independent of hepatic OSTb expression.
Our Ostb -/model made it possible to compare the consequence of OSTb deficiency and OSTa deficiency in mice but also to compare this to the few individuals described to date with SLC51A or SLC51B deficiency. In contrast to Asbt -/mice which show a similar malabsorptive phenotype as patients with an Asbt mutation, 15,38 Ostb -/mice do not reflect all characteristics of the 2 OSTb-deficient patients. The OSTb-deficient brothers suffer from congenital diarrhea and features of cholestasis, 17 whereas Osta -/and Ostb -/mice do not. Furthermore, the OSTa-deficient patient who was recently identified showed symptoms similar to the OSTb-deficient brothers, albeit with more severe signs of cholestasis. 18 While expression of Osta and Ostb is high in human livers, it is marginal in mouse livers under normal circumstances. 7 This may explain why OSTa-and OSTb-deficient patients experience liver histological changes and elevated liver enzymes ALT, AST and GGT, while there is only a trend towards a modest increase in ALT and ALP in the Ostb -/mice. Protective mechanisms are initiated in mice with OSTa or OSTb deficiency to reduce the bile acid load, which likely explains the ameliorated phenotype in older mice, 10,11,13,14 although the elongated small intestine remains present in aged Ostb -/mice. In addition, mice have a different gut microbiome composition and enzymatic bile acid (re)hydroxylation repertoire leading to a distinct bile acid composition and conjugation. 39 The mouse bile acid pool is less hydrophobic and toxic which may dampen liver damage and is also much reduced in OSTa-and OSTb-deficient mice, lowering the level of diarrhea despite the severely affected ileal morphology.
Gene expression of ileal Fgf15 was increased, inversely correlated with Asbt expression in the ileum and Cyp7a1 in the liver, implying accumulation of bile acids in the enterocyte and dampening of bile acid synthesis. Surprisingly, gene expression of Fabp6 was not elevated, however, conflicting results on gene expression of Fabp6 have been observed in Osta -/mice before. 11-13 Short-term inhibition of OSTa-OSTb in vivo leads to increased FXR activation in enterocytes 40 and it was previously demonstrated that the increase in Fgf15 expression in Osta -/mice is due to FXR activation. Recent evidence indicates that the ileal histological changes in Osta -/are secondary to enterocyte injury caused by bile acid accumulation, since disruption of Asbt in Osta -/mice restores the intestinal phenotype completely. 11 Even though expression of Asbt is partly downregulated, Osta -/and Ostb -/mice are not able to fully restore the ileal morphology, suggesting that bile acid accumulation in enterocytes is still present. Furthermore, expression of ileal Mrp3 is not increased, supporting the evidence that MRP3 does not have a major role in conjugated bile acid transport. 41 Finally, Osta -/and Ostb -/organoids do not show an altered phenotype, suggesting that bile acids cause the altered phenotype in the ileum.
In conclusion, OSTa-OSTb is an important heterodimeric bile acid transporter. Knockout of either Osta or Ostb results in a severe ileal phenotype that is in line with previous Osta knockout studies. During cholestasis, knockout of either Osta or Ostb seems to ameliorate liver damage. However, unlike in Osta -/mice, these beneficial effects are paralleled by an intestinal motility phenotype in Ostb -/mice, potentially contributing to a significantly lower survival rate and higher body weight loss. This is the first evidence that the role of OSTb differs from OSTa and suggests that OSTb might also have an additional, unidentified, intestinal function.