UBIAD1 Plays an Essential Role in the Survival of Pancreatic Acinar Cells

UbiA prenyltransferase domain-containing protein 1 (UBIAD1) is a vitamin K2 biosynthetic enzyme. We previously showed the lethality of this enzyme in UBIAD1 knockout mice during the embryonic stage. However, the biological effects of UBIAD1 deficiency after birth remain unclear. In the present study, we used a tamoxifen-inducible systemic UBIAD1 knockout mouse model to determine the role of UBIAD1 in adult mice. UBIAD1 knockout resulted in the death of the mice within about 60 days of administration of tamoxifen. The pancreas presented with the most prominent abnormality in the tamoxifen-induced UBIAD1 knockout mice. The pancreas was reduced remarkably in size; furthermore, the pancreatic acinar cells disappeared and were replaced by vacuoles. Further analysis revealed that the vacuoles were adipocytes. UBIAD1 deficiency in the pancreatic acinar cells caused an increase in oxidative stress and autophagy, leading to apoptotic cell death in the tamoxifen-induced UBIAD 1 knockout mice. These results indicate that UBIAD1 is essential for maintaining the survival of pancreatic acinar cells in the pancreas.


Introduction
Vitamin K is a cofactor for γ-glutamyl carboxylase (GGCX), an enzyme that converts specific glutamic acid residues in several substrate proteins involved in blood coagulation and bone metabolism to γ-carboxyglutamic acid residues [1,2]. In addition to its role as a cofactor for GGCX, vitamin K is involved in the transcriptional regulation of the nuclear receptor the steroid and xenobiotic sensing nuclear receptor (SXR) (also known as pregnane X receptor or PXR) [3][4][5] and regulates protein kinase A (PKA) signaling in osteoblasts and hepatocellular carcinoma cells [6]. Furthermore, menaquinone-4 (MK-4) is reported to have a role in the mitochondrial electron transport chain in Drosophila [7].
There are two naturally occurring forms of vitamin K, phylloquinone (PK or vitamin K 1 ) and the group of menaquinones (MKs). All forms of vitamin K are characterized by a common 2-methyl-1,4-naphthoquinone ring structure (menadione, MD) and a hydrophobic polyisoprenoid side chain attached at the 3-carbon position of the nucleus. PK has a monounsaturated side chain of four isoprenyl residues and is primarily found in leafy green vegetables. MKs can be classified into 14 types on the basis of the length of their unsaturated side chains. Among them, MK-4 is

The Pancreas of Tamoxifen-Induced UBIAD1-Deficient Mice was Markedly Atrophied
Female UBIAD1-cKO mice were analyzed because they seemed to survive for longer periods

The Pancreas of Tamoxifen-Induced UBIAD1-Deficient Mice Was Markedly Atrophied
Female UBIAD1-cKO mice were analyzed because they seemed to survive for longer periods than male mice.
Body and tissue weights of UBIAD1-cKO female mice were measured at 40 days after administration of tamoxifen and compared with those of the tamoxifen-treated CAG-Cre-ERT −/− UBIAD1 fl/fl (Flox) mice carrying conditional UBIAD1 alleles, but lacking Cre expression (Table 1). No significant differences in body weights were noted between the Flox and UBIAD1-cKO mice (both tamoxifen-treated and non-treated). However, the weight of the pancreas in the tamoxifen-treated UBIAD1-cKO mice was markedly reduced when compared with the Flox mice and the tamoxifen non-treated UBIAD1-cKO mice (Table 1). On the other hand, the weights of other tissues were not significantly different among the four types of mice (Table 1). MK-4 and deuterium-labeled MK-4 (MK-4-d 7 ) concentrations converted from deuterium-labeled MD (MD-d 8 ) in the tamoxifen-treated UBIAD1-cKO mice were significantly lower than in the tamoxifen-treated Flox mice (Table 2). Surprisingly, the pancreas of the tamoxifen-treated UBIAD1-cKO mice was markedly atrophied when compared with the tamoxifen-treated Flox mice, as shown in Figure 2A. Furthermore, UBIAD1 messenger RNA (mRNA) expression levels were markedly reduced in the tamoxifen-treated UBIAD1-cKO mice when compared with the tamoxifen-treated Flox mice and the tamoxifen non-treated UBIAD1-cKO mice ( Figure 2B). UBIAD1 mRNA levels in other tissues were also markedly reduced in the tamoxifen-treated UBIAD1-cKO mice ( Figure S2, Supplementary Materials).

Disappearance of Pancreatic Acinar Cells and Formation of Vacuoles in the Pancreas of Tamoxifen-Induced UBIAD1-Deficient Mice
The pancreas of the tamoxifen-treated UBIAD1-cKO mice was remarkably smaller. Tissue sections were prepared, and hematoxylin-eosin (H&E) staining was performed. Pancreatic acinar cells were absent in the tamoxifen-treated UBIAD1-cKO mice; instead, several vacuoles were noted ( Figure 3). On the other hand, the presence of internal α, β, and δ cells was confirmed by Gomori aldehyde fuchsin staining (Figure 4). No atrophy of the pancreatic islets of tamoxifen-treated UBIAD1-cKO mice was noted. The pancreas of tamoxifen-treated Flox mice was stained with insulin antibody only in the pancreatic islets, whereas, in tamoxifen-treated UBIAD1-cKO mice, pancreatic islets and portions other than the pancreatic islets were also widely stained ( Figure 4). These results indicate that insulin secreted from pancreatic islets is dispersed within the pancreas in tamoxifen-treated UBIAD1-cKO mice. *** p < 0.001 compared to corresponding value in Flox mice, Flox mice + Tam, and UBIAD1-cKO mice. Table 2. Menaquinone-4 (MK-4) and deuterium-labeled MK-4 (MK-4-d7) concentrations in the Flox and UBIAD1-cKO mice at 40 days after administration of tamoxifen.

Disappearance of Pancreatic Acinar Cells and Formation of Vacuoles in the Pancreas of Tamoxifen-Induced UBIAD1-Deficient Mice
The pancreas of the tamoxifen-treated UBIAD1-cKO mice was remarkably smaller. Tissue sections were prepared, and hematoxylin-eosin (H&E) staining was performed. Pancreatic acinar cells were absent in the tamoxifen-treated UBIAD1-cKO mice; instead, several vacuoles were noted ( Figure 3). On the other hand, the presence of internal ,  and  cells was confirmed by Gomori aldehyde fuchsin staining ( Figure 4). No atrophy of the pancreatic islets of tamoxifentreated UBIAD1-cKO mice was noted. The pancreas of tamoxifen-treated Flox mice was stained with insulin antibody only in the pancreatic islets, whereas, in tamoxifen-treated UBIAD1-cKO mice, pancreatic islets and portions other than the pancreatic islets were also widely stained ( Figure 4). These results indicate that insulin secreted from pancreatic islets is dispersed within the pancreas in tamoxifen-treated UBIAD1-cKO mice.     In the Gomori aldehyde fuchsin-stained sections, elastic fibers and pancreatic islet β class cells were stained purple, pancreatic islet δ cells were stained light green, pancreatic islet α cells were stained red, and connective tissue was stained green. Immunohistological staining with insulin specific antibody in the pancreas of the tamoxifen-treated Flox and UBIAD1-cKO mice at 40 days after administration of tamoxifen.

The Vacuoles Found in the Pancreas of Tamoxifen-Treated UBIAD1-cKO Mice Were Adipocytes
Immunohistochemical staining using adipocyte-specific antibodies (Fatty acid binding protein 4 (FABP4), Peroxisome proliferator-activated receptor (PPAR), and perilipin) was performed to investigate the vacuoles found in the pancreas of the tamoxifen-treated UBIAD1-cKO mice ( Figure 5). These results indicate that, in the pancreas of tamoxifen-treated UBIAD1-cKO mice, pancreatic acinar cells disappeared and adipocytes were formed at the disappeared site.

Pancreatic Acinar Cells in the Pancreas of Tamoxifen-Treated UBIAD1-cKO Mice Increased Oxidative Stress and Autophagy and Disappeared by Apoptosis
In order to determine the cause of the formation of vacuoles and the disappearance of pancreatic acinar cells in the cKO mice, we investigated the involvement of oxidative stress, autophagy, and apoptosis. Strong 4-Hydroxy nonenal (4-HNE)-positive staining observed at the site where the acinar cells disappeared indicated that the pancreatic acinar cells in tamoxifen-treated UBIAD1-cKO mice were exposed to strong oxidative stress. In addition, positive staining for Microtubule-associated protein light chain 3 (LC3), a central protein in the autophagy pathway, was detected in atrophied pancreatic acinar cells in the tamoxifen-treated UBIAD1-cKO mice. Furthermore, cleaved caspase-3 positive cells were observed in the atrophic and non-atrophic pancreatic acinar cells ( Figure 6). These findings indicated that pancreatic acinar cells in the tamoxifen-treated UBIAD1-cKO mice disappeared via apoptosis due to oxidative stress and enhancement of autophagy.
Immunohistochemical staining using adipocyte-specific antibodies (Fatty acid binding protein 4 (FABP4), Peroxisome proliferator-activated receptor (PPAR), and perilipin) was performed to investigate the vacuoles found in the pancreas of the tamoxifen-treated UBIAD1-cKO mice ( Figure 5). These results indicate that, in the pancreas of tamoxifen-treated UBIAD1-cKO mice, pancreatic acinar cells disappeared and adipocytes were formed at the disappeared site.

Figure 5.
Expression of adipocyte-specific markers in the vacuoles in tamoxifen-treated UBIAD1-cKO mice. Immunohistological staining with FABP4, PPAR, and perilipin in the pancreas of the tamoxifen-treated Flox and UBIAD1-cKO mice at 40 days after administration of tamoxifen. The tissues of immunohistological staining with PPAR staining were counterstained with hematoxylin.

Pancreatic Acinar Cells in the Pancreas of Tamoxifen-Treated UBIAD1-cKO Mice increased Oxidative Stress and Autophagy and Disappeared by Apoptosis
In order to determine the cause of the formation of vacuoles and the disappearance of pancreatic acinar cells in the cKO mice, we investigated the involvement of oxidative stress, autophagy, and apoptosis. Strong 4-Hydroxy nonenal (4-HNE)-positive staining observed at the site where the acinar cells disappeared indicated that the pancreatic acinar cells in tamoxifentreated UBIAD1-cKO mice were exposed to strong oxidative stress. In addition, positive staining for Microtubule-associated protein light chain 3 (LC3), a central protein in the autophagy pathway, was detected in atrophied pancreatic acinar cells in the tamoxifen-treated UBIAD1-cKO mice. Furthermore, cleaved caspase-3 positive cells were observed in the atrophic and nonatrophic pancreatic acinar cells ( Figure 6). These findings indicated that pancreatic acinar cells in the tamoxifen-treated UBIAD1-cKO mice disappeared via apoptosis due to oxidative stress and enhancement of autophagy.

Abundance of Neutrophils and Mesenchymal Stem Cells in the Pancreas of Tamoxifen-Treated UBIAD1-cKO Mice
The pathology of the pancreas in tamoxifen-treated UBIAD1-cKO mice is similar to that of chronic pancreatitis. Several cells positively stained for myeloperoxidase (MPO), a neutrophil marker, were detected in the pancreas of tamoxifen-treated UBIAD1-cKO mice ( Figure 7). Furthermore, in order to investigate whether undifferentiated mesenchymal stem cells were differentiated into adipocytes in the pancreatic tissue, the cells were stained with an antibody against CD105 (as known as Endoglin), a mesenchymal stem cell marker. As a result, several CD105-positive cells were detected in the pancreas of the tamoxifen-treated UBIAD1-cKO mice (Figure 7). These results suggest that the mesenchymal stem cells may have invaded during inflammation and differentiated into adipocytes in the pancreas of these mice.

Abundance of Neutrophils and Mesenchymal Stem Cells in the Pancreas of Tamoxifen-Treated UBIAD1-cKO Mice
The pathology of the pancreas in tamoxifen-treated UBIAD1-cKO mice is similar to that of chronic pancreatitis. Several cells positively stained for myeloperoxidase (MPO), a neutrophil marker, were detected in the pancreas of tamoxifen-treated UBIAD1-cKO mice ( Figure 7). Furthermore, in order to investigate whether undifferentiated mesenchymal stem cells were differentiated into adipocytes in the pancreatic tissue, the cells were stained with an antibody against CD105 (as known as Endoglin), a mesenchymal stem cell marker. As a result, several CD105-positive cells were detected in the pancreas of the tamoxifen-treated UBIAD1-cKO mice (Figure 7). These results suggest that the mesenchymal stem cells may have invaded during inflammation and differentiated into adipocytes in the pancreas of these mice.

Serum Glucose and Insulin Concentrations and Glucose Tolerance in Tamoxifen-Induced UBIAD1-Deficient Mice
We measured the serum glucose and insulin concentrations in the Flox and UBIAD1-cKO mice every week following tamoxifen administration. Serum glucose concentration was significantly lowered in tamoxifen-treated UBIAD1-cKO mice when compared with the tamoxifen-treated Flox mice; likewise, the concentration of serum insulin in these mice tended to be lower than that in the Flox mice ( Figure 8A,B). Therefore, we conducted a glucose tolerance test in the two types of mice 40 days after tamoxifen administration. The blood glucose level in the tamoxifen-treated UBIAD1-cKO mice at 0 min before glucose loading was significantly lower than that of the tamoxifen-treated Flox mice. A slight increase in the level was noted in the tamoxifen-treated UBIAD1-cKO mice after glucose loading; however, they remained significantly lower in these mice when compared with the tamoxifen-treated Flox mice ( Figure  8C) indicating that tamoxifen-induced UBIAD1 deficient mice may have decreased glucose absorption capacity and pancreatic function.

Serum Glucose and Insulin Concentrations and Glucose Tolerance in Tamoxifen-Induced UBIAD1-Deficient Mice
We measured the serum glucose and insulin concentrations in the Flox and UBIAD1-cKO mice every week following tamoxifen administration. Serum glucose concentration was significantly lowered in tamoxifen-treated UBIAD1-cKO mice when compared with the tamoxifen-treated Flox mice; likewise, the concentration of serum insulin in these mice tended to be lower than that in the Flox mice ( Figure 8A,B). Therefore, we conducted a glucose tolerance test in the two types of mice 40 days after tamoxifen administration. The blood glucose level in the tamoxifen-treated UBIAD1-cKO mice at 0 min before glucose loading was significantly lower than that of the tamoxifen-treated Flox mice. A slight increase in the level was noted in the tamoxifen-treated UBIAD1-cKO mice after glucose loading; however, they remained significantly lower in these mice when compared with the tamoxifen-treated Flox mice ( Figure 8C) indicating that tamoxifen-induced UBIAD1 deficient mice may have decreased glucose absorption capacity and pancreatic function.

Abnormal Tissue Structure and Decreased Sodium-Dependent Glucose Transporter 1 (SGLT1) and Glucose Transporter 2 (GLUT2) Expressions in the Duodenum of Tamoxifen-Treated UBIAD1-cKO Mice
UBIAD1 is deficient not only in the pancreas but also in the duodenum of the tamoxifentreated UBIAD1-cKO mice, thereby affecting the glucose absorption capacity of the duodenum. The basolateral membrane in the duodenum of the tamoxifen-treated UBIAD1-cKO mice appeared disorganized, while the surface of intestinal villus was rough when compared with those in the tamoxifen-treated Flox mice (Figure 9). Glucose is absorbed through the intestine by a transepithelial transport system that is initiated at the apical membrane by the co-transporter SGLT1; intracellular glucose is then assumed to diffuse across the basolateral membrane through GLUT2. Marked SGLT1 and GLUT2 expressions were observed on the luminal side of the villi in the duodenum of the tamoxifen-treated Flox mice, but not in the tamoxifen-treated UBIAD1-cKO mice (Figure 9). Expression of SGLT1 and GLUT2 was very weak in duodenal epithelial cells of the tamoxifen-treated UBIAD1-cKO mice.

Abnormal Tissue Structure and Decreased Sodium-Dependent Glucose Transporter 1 (SGLT1) and Glucose Transporter 2 (GLUT2) Expressions in the Duodenum of Tamoxifen-Treated UBIAD1-cKO Mice
UBIAD1 is deficient not only in the pancreas but also in the duodenum of the tamoxifen-treated UBIAD1-cKO mice, thereby affecting the glucose absorption capacity of the duodenum. The basolateral membrane in the duodenum of the tamoxifen-treated UBIAD1-cKO mice appeared disorganized, while the surface of intestinal villus was rough when compared with those in the tamoxifen-treated Flox mice (Figure 9). Glucose is absorbed through the intestine by a transepithelial transport system that is initiated at the apical membrane by the co-transporter SGLT1; intracellular glucose is then assumed to diffuse across the basolateral membrane through GLUT2. Marked SGLT1 and GLUT2 expressions were observed on the luminal side of the villi in the duodenum of the tamoxifen-treated Flox mice, but not in the tamoxifen-treated UBIAD1-cKO mice (Figure 9). Expression of SGLT1 and GLUT2 was very weak in duodenal epithelial cells of the tamoxifen-treated UBIAD1-cKO mice.

Discussion
UBIAD1 is an essential enzyme required for MK-4 biosynthesis in mice and humans. The conventional UBIAD1 knockout mice do not survive during the early gestation period, thus indicating that UBIAD1 plays an essential role in the developmental process [14]. UBIAD1 exists in all tissues, and is expected to maintain the function of each tissue by biosynthesizing MK-4; nonetheless, the significance of this biosynthesis in each organization remains unclear. Therefore, in order to clarify the importance and role of UBIAD1 in each tissue in mice after birth, we generated a tamoxifen-induced systemic UBIAD1-deficient mice model and evaluated the effects of UBIAD1 deficiency in these mice after maturation.
In the present study, the UBIAD1-deficient mice did not survive after maturation. Female and male UBIAD1-cKO mice demonstrated similar rates of death after administration of tamoxifen, but male UBIAD1-cKO mice died earlier than the female mice. Previously, we reported that the concentration of MK-4 in tissues of male wild-type mice was lower than that of the female wild-type mice [9]. Hence, it was considered that male UBIAD1-cKO mice in which UBIAD1 deficiency was induced by tamoxifen administration became MK-4-deficient earlier than female UBIAD1-cKO mice, leading to lethality. The body and tissue weights of female UBIAD1-cKO mice were measured 40 days after induction of UBIAD1 deficiency by tamoxifen. No significant differences in body weight were noted between the UBIAD1-cKO and Flox mice treated with tamoxifen, whereas the weight of the pancreas in the UBIAD1-cKO mice was significantly lower than that in the Flox mice. The pancreas of tamoxifen-treated UBIAD1-cKO mice was markedly atrophied and reduced in size; additionally, MK-4 concentration and UBIAD1 expression were decreased in the whole tissues of these mice. It is known that the concentration of MK-4 is highest in the pancreas when compared with any other tissue [9]. In a previous study, Thomas et al. indicated that MK-4 is detected in pancreatic secretions and is present within the secretory pathway of acinar cells, thereby suggesting that MK-4 has a unique function in acinar cells [15]. Consistent with the low concentrations of phylloquinone reported in

Discussion
UBIAD1 is an essential enzyme required for MK-4 biosynthesis in mice and humans. The conventional UBIAD1 knockout mice do not survive during the early gestation period, thus indicating that UBIAD1 plays an essential role in the developmental process [14]. UBIAD1 exists in all tissues, and is expected to maintain the function of each tissue by biosynthesizing MK-4; nonetheless, the significance of this biosynthesis in each organization remains unclear. Therefore, in order to clarify the importance and role of UBIAD1 in each tissue in mice after birth, we generated a tamoxifen-induced systemic UBIAD1-deficient mice model and evaluated the effects of UBIAD1 deficiency in these mice after maturation.
In the present study, the UBIAD1-deficient mice did not survive after maturation. Female and male UBIAD1-cKO mice demonstrated similar rates of death after administration of tamoxifen, but male UBIAD1-cKO mice died earlier than the female mice. Previously, we reported that the concentration of MK-4 in tissues of male wild-type mice was lower than that of the female wild-type mice [9]. Hence, it was considered that male UBIAD1-cKO mice in which UBIAD1 deficiency was induced by tamoxifen administration became MK-4-deficient earlier than female UBIAD1-cKO mice, leading to lethality. The body and tissue weights of female UBIAD1-cKO mice were measured 40 days after induction of UBIAD1 deficiency by tamoxifen. No significant differences in body weight were noted between the UBIAD1-cKO and Flox mice treated with tamoxifen, whereas the weight of the pancreas in the UBIAD1-cKO mice was significantly lower than that in the Flox mice. The pancreas of tamoxifen-treated UBIAD1-cKO mice was markedly atrophied and reduced in size; additionally, MK-4 concentration and UBIAD1 expression were decreased in the whole tissues of these mice. It is known that the concentration of MK-4 is highest in the pancreas when compared with any other tissue [9]. In a previous study, Thomas et al. indicated that MK-4 is detected in pancreatic secretions and is present within the secretory pathway of acinar cells, thereby suggesting that MK-4 has a unique function in acinar cells [15]. Consistent with the low concentrations of phylloquinone reported in exocrine tissues [16,17], no vitamin K compounds other than MK-4 were detected in pancreatic secretions. Thomas et al. reported that the detection of MK-4 in pancreatic secretions indicates its presence in the secretory pathway of the acinar cells [15]. Although the reason for the abundance of MK-4 in the pancreas is not clear, the findings of the current study suggest that it may play an important role in this organ.
H&E staining revealed an absence of pancreatic acinar cells and an increase in the number of vacuoles in the tissues of the tamoxifen-treated UBIAD1-cKO mice. The number of pancreatic islets was similar in the tamoxifen-treated UBIAD1-cKO mice and Flox mice, indicating that the abnormality was limited to the pancreatic acinar cells. The pancreas of the tamoxifen-treated UBIAD1-cKO mice was remarkably reduced in size. However, no atrophy of the pancreatic islets of tamoxifen-treated UBIAD1-cKO mice was noted. We considered that the cause of the atrophy of the pancreas of UBIAD1-cKO mice was the loss of pancreatic acinar cells that occupy 90% of the pancreas. Although various digestive enzymes are present in pancreatic acinar cells, it is speculated that UBIAD1 deficiency did not cause significant cell damage in pancreas islets because pancreatic islets do not produce the digestive enzymes. As a result of observing the expression of insulin by immunohistological staining, the insulin in pancreatic islets was stained equally in both tamoxifen-treated Flox mice and UBIAD1-cKO mice. From this, it is speculated that the level of insulin secretion in pancreatic islets is not reduced in cKO mice. However, no insulin was observed in the pancreatic acinar cells of the Flox mice, whereas, in the UBIAD1-cKO mice, extensive insulin diffusion to the site of the pancreatic acinar cells was noted. The pancreatic acinar cells in the tamoxifen-treated UBIAD1-cKO mice almost disappeared, and most of the cells were replaced by vacuoles. Interestingly, the vacuoles stained positively for the adipocyte marker proteins FABP4, PPAR [18,19], and perilipin [20,21], indicating that the pancreatic acinar cells were replaced by fat cells. Although the functions of UBIAD1 and MK-4 in the pancreas are not clear, periostin, a member of a vitamin K-dependent γ-carboxylated protein family, is reported to be involved in pancreatitis [22]. In normal mice, periostin expression is strongly induced throughout the fibrotic reaction in cerulein-mediated acute pancreatitis [23]. However, in periostin-deficient mice, the most striking phenotype of periostin-knockout mice is the replacement of acinar cells by adipocytes after induction of acute pancreatitis [23]. The involvement of periostin was not further examined in the current study. However, MK-4 deficiency is caused by UBIAD1 deficiency in tamoxifen-induced UBIAD1-cKO mice, and since periostin is not γ-carboxylated by GGCX, it is speculated that the acinar cells may have disappeared and been replaced with adipocytes.
In humans and mice, it is reported that vacuoles are formed at the site of pancreatic acinar cells during acute pancreatitis [24]. Pancreatic acinar cells are the functional units of the exocrine pancreas. They are known to synthesize, store, and secrete digestive enzymes. Under normal physiological conditions, digestive enzymes are activated only after they reach the duodenum [25]. Premature activation of these enzymes within pancreatic acinar cells leads to the onset of acute pancreatitis [25], wherein the acinar cells are digested by the enzymes. The causes of acute pancreatitis include oxidative stress and increased autophagy. Oxidative stress and its constant companion, inflammation, play critical roles in the pathogenesis of pancreatitis and its numerous complications [26][27][28][29]. Oxidative stress is caused by a combination of the increased production of reactive oxygen species and impaired antioxidant capacity. Previously, it was reported that autophagy is induced by the supramaximal stimulation of cerulein, and is directly related to trypsinogen activation and the onset of acute pancreatitis in mice [30]. Therefore, we investigated the involvement of oxidative stress, autophagy, and apoptosis in pancreatic acinar cell loss in these mice. The atrophied pancreatic acinar cells in the tamoxifen-treated UBIAD1-cKO mice were positive for the oxidative stress marker 4-HNE [31], and a large amount of LC3 deposition (indicating autophagy) was detected. Furthermore, the pancreatic acinar cells in the tamoxifen-treated UBIAD1-cKO mice were positive for the cleaved caspase-3 apoptotic marker. These results suggested that the pancreatic acinar cells disappeared via apoptosis due to enhanced oxidative stress and autophagy. Previous reports suggested that MK-4 prevents oxidative cell death in developing oligodendrocytes and neurons [32,33], and that UBIAD1 prevents oxidative damage in cardiovascular tissues [34], and sustains mitochondrial function in Drosophila [7]. Since both MK-4 and UBIAD1 are thought to be involved in anti-oxidation, the deficiency of both UBIAD1 and MK-4 may result in oxidative stress, autophagy enhancement, and apoptosis in pancreatic acinar cells. Mesenchymal stem cells were shown to differentiate into adipocytes [35]. However, according to the present study, most of the pancreatic acinar cells disappeared probably via apoptosis due to oxidative stress and autophagy; moreover, CD105-positive mesenchymal stem cells were frequently found at the site of these cells. Taken together, these results indicate that, after the disappearance of the pancreatic acinar cells in the tamoxifen-treated UBIAD1-cKO mice, mesenchymal stem cells may have invaded the tissues and differentiated into adipocytes due to stimulation via insulin in the pancreas. Thus, UBIAD1 and MK-4 may play essential roles in the survival and function of pancreatic acinar cells.
Serum glucose concentration was significantly lower in the UBIAD1-cKO mice than in the Flox mice after tamoxifen administration, and both before and after the oral glucose tolerance test (OGTT). This may be due to a decrease in pancreatic function. However, analysis of the duodenal tissue revealed a disordered basolateral membrane in the tamoxifen-treated UBIAD1-cKO mice, and the surface of intestinal villus was rough. Moreover, SGLT1 and GLUT2 expression levels were decreased in the duodenum of the tamoxifen-treated UBIAD1-cKO mice. In the duodenum of tamoxifen-treated UBIAD1-cKO mice, UBIAD1 deficiency is thought to cause abnormalities in intestinal epithelial cell differentiation and maturation. As a result, expression of SGLT1 and GLUT2 in intestinal epithelial cells may be reduced. Intestinal glucose absorption is mediated by SGLT1, but GLUT2 is thought to provide a basolateral outlet. GLUT2 can be recruited to the apical membrane after application of a high-glucose bolus into the lumen to allow for the bulk absorption of glucose via facilitated diffusion. Furthermore, SGLT1 and GLUT2 are suggested to play important roles in intestinal glucose sensing [36][37][38][39]. The decrease in the expression levels of SGLT1 and GLUT2 is the main factor responsible for the reduction in glucose absorption from the intestine in UBIAD1-deficient mice. Thus, UBIAD1 is thought to be necessary for cell proliferation and differentiation, and for maintenance of the function of the intestinal epithelial cells. Previous reports demonstrated a relationship between vitamin K intake and insulin sensitivity or glucose tolerance [40]. However, the action of MK-4 on intestinal glucose absorption remains to be shown. The results of the current study suggest that UBIAD1 and MK-4 play important roles in maintaining the morphology and function of intestinal epithelial cells and the expression levels of SGLT1 and GLUT2, which are important for glucose absorption. Further detailed analyses are required to examine this phenomenon in the future.
In summary, our data demonstrate that the loss of UBIAD1 in adult mice results in decreased survival, particularly due to severe abnormalities in the pancreas. The pathology of the UBIAD1-deficient pancreas is similar to that of acute pancreatitis, resulting in the disappearance of pancreatic acinar cells, which are replaced by adipocytes. The pancreatic acinar cells in UBIAD1-deficient mice demonstrated increased oxidative stress, autophagy, and apoptosis. Furthermore, mesenchymal stem cells in the pancreas may have been exposed to pancreatic insulin and differentiated into adipocytes. These findings indicate that both UBIAD1 and MK-4 play essential roles in maintaining the survival of pancreatic acinar cells.

Ethics Statement
All animal experimental protocols were performed in accordance with the Guidelines for Animal Experiments at Kobe Pharmaceutical University and were approved by Kobe Pharmaceutical University Committee for Animal Care and Use, Kobe, Japan (2016-001; 1 April 2016, 2017-006; 1 April 2017, 2018-055; 1 April 2018). All surgical procedures were performed under isoflurane anesthesia, and all efforts were made to minimize suffering.

Real-Time PCR
Total RNA was isolated from mouse tissue using Isogen (Nippon Gene, Tokyo, Japan) according to the manufacturer's protocol. First-strand complementary DNA (cDNA) synthesis was performed using ReverTra Ace qPCR RT Master Mix with genomic DNA (gDNA) Remover (TOYOBO, Tokyo, Japan). The cDNA was mixed with SsoAdvanced Universal SYBR Green Supermix (Bio-Rad, Hercules, CA, USA), and amplified using the CFX96 real-time PCR system (Bio-Rad). Mouse  The mice were administered MD-d 8 orally as a single dose (10 µmol/kg body weight). After 24 h, the animals were sacrificed, and tissues were removed and stored at −80 • C for analysis. MK-4, MK-4 epoxide, MK-4-d 7 , and MK-4-d 7 epoxide levels were measured by LC-APCI-MS/MS as reported previously [8,21].