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Impaired fatty acid utilization in thioredoxin binding protein-2 (TBP-2)-deficient mice: a unique animal model of Reye syndrome

Shin-ichi Oka^,**, Wenrui Liu, Hiroshi Masutani, Hiromi Hirata¹, Yoichi Shinkai, Shu-ichi Yamada^¶,2, Toru Yoshida, Hajime Nakamura and Junji Yodoi^,3

Laboratory of Infection and Prevention, Department of Biological Response,
Laboratory of Growth Regulation,
^¶ Laboratory of Signal Transduction, Department of Cell Biology,
Laboratory of Mouse Model, Experimental Research Center for Infectious Diseases, Institute for Virus Research, Kyoto University, Kawahara-cho, Sakyo, Kyoto; Department of Experimental Therapeutics, Translational Research Center, Kyoto University Hospital, Kawahara-cho, Sakyo-ku, Kyoto, Japan;
^** Cell Dynamics Research Group, Research Institute for Cell Engineering, National Institute of Advanced Industrial Science and Technology (AIST), Midorigaoka, Ikeda, Osaka

³Correspondence: Department of Biological Responses, Institute for Virus Research, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo, Kyoto 606-8507, Japan. E-mail: yodoi{at}virus.kyoto-u.ac.jp

SPECIFIC AIM

Thioredoxin binding protein-2 (TBP-2) is a negative regulator of thioredoxin and implicated to play a critical role in glucose and lipid metabolism. The aim of our study was to investigate the in vivo role of Thioredoxin Binding Protein-2 (TBP-2) by complete disruption of TBP-2 in mice (TBP-2^–/– mice).

PRINCIPAL FINDINGS

1. TBP-2^–/– mice are predisposed to death with severe bleeding during fasting
We have generated mice with targeted inactivation of TBP-2 (TBP-2^–/– mice). These mice were viable, fertile, and showed no gross appearance of abnormalities in adulthood. In contrast, TBP-2^–/– mice are predisposed to death under fasting conditions (Fig. 1 A). After wild-type 72 h fasting, several TBP-2^–/– mice could not be rescued by refeeding (Fig. 1A ). Before death, TBP-2^–/– mice exhibited hematuria (Fig. 1B ). Overall, the gastrointestinal region had a dark red appearance, suggesting that gastrointestinal bleeding was occurring in these mice (Fig. 1C ). The bleeding tendency was not observed in TBP-2^–/– mice during regular feeding (Fig. 1B, C ). These results indicate that TBP-2 deficiency leads to a bleeding tendency under fasting conditions. As shown in Fig. 1D , TBP-2 expression was significantly up-regulated in the liver, heart, and lungs in response to fasting, suggesting that TBP-2 is a fasting response gene and that augmented TBP-2 has an important role in the prevention of bleeding under fasting conditions.

View larger version (62K): [in this window] [in a new window]   Figure 1. TBP-2–/– mice showed a predisposition for death with severe bleeding during fasting. A) Decreased survival rate of TBP-2–/– mice in a fasted state. TBP-2–/– mice were subjected to fasting, while water continued to be freely available. The percentage of surviving animals is indicated as a function of time. The number of mice (n) in each group is as indicated. B) Hematuria in fasted TBP-2–/– mice. TBP-2–/– mice displayed hematuria in the bladder after 48 h fasting. C) Gastrointestinal bleeding in fasted TBP-2–/– mice. Mice of each genotype were subjected to fasting, and the gastrointestines were dissected. D) Fasting-induced TBP-2 expression. Northern blot analyses were performed using total RNA in the liver (top panel), heart (middle panel), and lung (bottom panel) from fed or fasted mice.

2. TBP-2^–/– mice exhibited coagulation dysregulation under fasting conditions
Prothrombin time and activated partial thromboplastin time did not differ between wild-type and TBP-2^–/– mice during feeding but were significantly prolonged in TBP-2^–/– mice during fasting. Anti-thrombin III activity was reduced in fasted TBP-2^–/– mice compared with wild-type mice. Neither platelet counts nor plasma fibrinogen levels were significantly changed in TBP-2^–/– mice compared with wild-type mice, suggesting that the hemorrhage did not result from disseminated intravascular coagulation.

3. Liver steatosis and multi-organ dysfunction occur in fasted TBP-2^–/– mice
The liver in TBP-2^–/– mice was yellow during fasting. Histological studies showed microvesicular and macrovesicular steatosis in TBP-2^–/– mice after 24 h fasting. The fatty liver was also confirmed by staining using Oil Red O. Levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH) were elevated in TBP-2^–/– mice during fasting compared with wild-type mice. The levels of blood urea nitrogen (BUN) also increased in TBP-2^–/– mice compared with wild-type mice and TBP-2^–/– mice exhibited both hyponatremia and hyperkalemia during fasting. Thus, renal failure was induced in TBP-2^–/– mice during fasting. These results suggested that hemorrhage as well as multi-organ failure occurs in fasted TBP-2^–/– mice.

4. Glucose supplementation corrected the fatal anomaly in TBP-2^–/– mice, while fatty acids were unable to do so
The anomaly observed in TBP-2^–/– mice strongly indicates the occurrence of nutritional dysmetabolism. To examine this hypothesis, these mice were housed with free access to 20% glucose or 10% oleic acid in drinking water, while deprived of other foods. With 20% glucose available in drinking water, TBP-2^–/– mice survived for 72 h, whereas TBP-2^–/– mice were not rescued with 10% oleic acid (Fig. 2 A). Wild-type mice were able to survive under both conditions. Glucose supplementation completely blocked the dysfunction of hepatocyte and renal cells in TBP-2^–/– mice (Fig. 2B ), and hemorrhage and fatty liver were also prevented by glucose supplementation. In contrast, TBP-2^–/– mice with oleic acid supplementation showed identical symptoms to fasted TBP-2^–/– mice. These results suggested that the fatal phenotype is triggered by glucose-deprivation, due to impaired fatty acid utilization in TBP-2^–/– mice.

View larger version (27K): [in this window] [in a new window]   Figure 2. TBP-2 is essential for fatty acid utilization but not for glucose utilization. A) Survival rate of TBP-2–/– mice with access to glucose or oleic acid in drinking water. TBP-2–/– mice were subjected to food deprivation with free access to 20% glucose or 10% oleic acid in drinking water. The percentage of surviving animals is indicated as a function of time. The number of mice (n) in each group is as indicated. B) Serum analyses in mice with access to 20% glucose. Mice of each genotype were housed with access to 20% glucose in drinking water for 72 h, and serum analyses were then performed. AST, alanine aminotransferase; ALT, aspartate aminotransferase; BUN, blood urea nitrogen; LDH, lactate dehydrogenase; Na, sodium; K, potassium. C–K) Abnormalities of serum glucose, insulin, lipid derivatives, ketone bodies, pyruvate and lactate in fasted TBP-2–/– mice. Serum samples from TBP-2–/– mice were collected after 24 h fasting, and the levels of glucose (C), insulin (D), free fatty acids (E), triglyceride (F), total cholesterol (G), phospholipids (H), ketone bodies (I), pyruvate (J), and lactate (K) were measured.

The levels of glucose were decreased in TBP-2^–/– mice under both feeding and fasting states (Fig. 2C ), suggesting that TBP-2^–/– mice preferentially use glucose as an energy source. Insulin levels were not significantly changed on feeding, whereas fasting-induced reduction of insulin was not observed in TBP-2^–/– mice (Fig. 2D ). Thus, paradoxical hyperinsulinemia is induced in TBP-2^–/– mice.

As shown in Fig. 2E , serum FFA levels were elevated in TBP-2^–/+ and TBP-2^–/– mice in a feeding state, and increased in TBP-2^–/– mice under fasting conditions. Thus, deposition of FFA is sharply correlated with TBP-2 deficiency.

The levels of serum triglyceride were slightly higher in TBP-2^–/+ and TBP-2^–/– mice in a feeding state, and significantly elevated in fasted TBP-2^–/– mice (Fig. 2F ). Levels of total cholesterol and phospholipids were increased in TBP-2^–/– mice during fasting (Fig. 2G, H ). Taken together, these observations suggested that deterioration of fatty acid utilization is a primary change that occurs with TBP-2 deficiency.

5. Acetyl-CoA catabolism is dysregulated in TBP-2^–/– mice
Ketone bodies were found to accumulate in TBP-2^–/– mice during feeding compared with wild-type mice and were further elevated upon fasting (Fig. 2I ). These results suggest that ß-oxidation is unimpaired, but that acetyl-CoA consumption is reduced in TBP-2^–/– mice. As shown in Fig. 2J, K , pyruvate and lactate were elevated in TBP-2^–/– mice during feeding. Although these metabolites decreased in fasted TBP-2^–/– mice, this is almost certainly due to the reduction of glucose levels. Overall, these results suggested that Krebs cycle-mediated acetyl-CoA consumption is reduced in TBP-2^–/– mice.

CONCLUSIONS AND SIGNIFICANCE

We have generated TBP-2 null mice to characterize TBP-2 function in vivo. Under normal housing conditions, these mice are viable and fertile, but under fasting conditions, their survival rate was sharply reduced, concomitant with severe bleeding, dyslipidemia, fatty liver, hypoglycemia, and hepatic and renal dysfunction. Our results suggest that disruption of TBP-2 reduces acetyl-CoA consumption, which serially leads to dysregulation of lipid and glucose metabolism, hepatocyte and renal failure, and coagulation dysfunction (Fig. 3 ). These abnormalities mimic the pathology of human fatty acid utilization deficient disorders such as Reye (-like) syndrome. These disorders are potentially fatal disorders, but the pathophysiology remains to be elucidated. It is largely unknown that impairment of fatty acids metabolism leads to coagulation dysfunction. Further investigation of the bleeding mechanism in fasted TBP-2^–/– mice might elucidate the link between energy metabolism and blood coagulation. The TBP-2^–/– mouse may represent an animal model that could be a useful tool for the study of Reye(-like) syndrome and the evaluation of therapeutic approaches for use in humans.

View larger version (21K): [in this window] [in a new window]   Figure 3. Schematic diagram of the abnormalities in TBP-2–/– mouse.

FOOTNOTES

¹ Present address: Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-Ku, Nagoya, Japan.

² Present address: Animal Research Laboratory, Bioscience Research-Education Center (BREC), Akita University, Hondo 1-1-1, Akita, 010-8543, Japan.

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4439fje;

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