Director

Toshiyuki Takai, PhD

Professor

Tokuo Yamamoto, PhD

Assistant Professor

Ju-Ryoun Soh, PhD

The center for advanced genome research, founded in 2004, is a research facility of the IDAC for the promotion of post-genome studies including genomic informatics, proteomics and systematic biology . Our mission is to investigate aging-related diseases including atherosclerosis, diabetes and obesity and develop biological strategies against these diseases. Our final goal is to establish therapeutic strategies for these aging-related diseases by stem cell biology and molecular targets against key molecules responsible for these common diseases.

We have been studying the metabolism of plasma lipoproteins that are stringently mediated by their receptors. Disorder of plasma lipoprotein metabolism is pathophysiologically linked with various common diseases including hyperlipidemia, atherosclerosis, diabetes and Alzheimer’s disease. There are several lipoprotein receptors including low density lipoprotein receptor (LDLR), very low density lipoprotein receptor (VLDLR), apolipoprotein E receptor 2 (apoER2) and the class B type I scavenger receptor (SRBI). Among these receptors, LDLR is one of the most well characterized lipoprotein receptors playing a key role in the homeostasis of cholesterol.

The genetic defects in the LDLR gene cause familial hypercholesterolemia, one of the most common genetic diseases in humans. VLDLR and apoER2 consist of five functional domains that resemble LDLR. Although non-mammalian VLDLR was shown to play a pivotal role in the oogenesis by supplying yolk precursors, the mammalian roles of VLDLR and apoER2 remain unclarified. LDLR is expressed in various tissues including the liver, while VLDLR and apoER2 are almost completely absent in the liver. Although LDLR plays a key role in the hepatic clearance of cholesterol-carrying LDL, the presence of other hepatic apoE-specific receptors has long been suggested. By characterizing cDNAs containing the ligand binding motifs common to the LDLR family, we showed that one of LDLR-related proteins (LRPs), designated as LRP5, is abundant in the liver and is able to bind apoE.

To evaluate the in vivo roles of LRP5, we generated mice carrying a mutated LRP5 gene. In contrast to the severe developmental defects of LRP6 mutant mice, LRP5-/- mice of both sexes developed and appeared normal, gaining weight at a rate equal to that of LRP5+/+ mice and were normally fertile. Under light microscopic examination of LRP5-/- mouse tissues, there were no apparent histological abnormalities in the tissues examined, including brain, kidney, liver, and pancreas.

The plasma levels of cholesterol in LRP5+/- and LRP5-/- mice fed a standard laboratory chow were identical to those of LRP5+/+ littermates. In contrast, when mice were fed a high-fat diet, plasma cholesterol levels were significantly increased both in LRP5+/- andLRP5-/- mice.

To determine the effects of LRP5 deficiency on the plasma clearance of chylomicron remnants, we injected fluorescent-labeled chylomicron remnants into LRP5-/- mice and +/+ littermates fed a high-fat diet. Approximately half of the injected chylomicron remnants were cleared from the plasma of LRP5+/+ mice at 30 min after injection, whereas over 80% remained in the plasma of LRP5-/- mice (Fig.1, left panel). Consistent with the delayed clearance, hepatic uptake of the injected fluorescence was markedly reduced in LRP5-/- mice (Fig. 1, right panel). These data indicate that LRP5 recognizes apoE-containing lipoproteins in vivo and plays a role in the hepatic clearance of chylomicron remnants.

Fig. 1
We also analyzed the effects of LRP5 deficiency on glucose metabolism using LRP5-/-, +/- and +/+ mice fed a normal laboratory chow diet. Although fasted blood glucose and insulin levels in LRP5-/- and +/- mice appeared identical to their +/+ littermates, LRP5-/- and LRP5+/- mice exhibited markedly impaired glucose tolerance (IGT) during an intraperitoneal glucose-tolerance test (Fig.2, left panel). Consistent with this marked glucose intolerance, the glucose-induced increase in plasma insulin concentration was lower in both LRP5-/- and LRP5+/- mice than in LRP5+/+ mice (Fig. 2, right panel).

Fig.2
Consistent with the glucose-tolerance test, the insulin secretary response to glucose in LRP5-/- islets was profoundly lower than that of LRP5+/+ islets, particularly at higher concentrations. To determine the involvement of Wnt proteins in glucose-induced insulin secretion, we pretreated LRP5+/+ islets with conditioned media (CM) from Wnt-3a, Wnt-5a or parental vector transfected L cells (neo-CM). The pretreatment of LRP5+/+ islets with Wnt-3a and -5a-CM for 16 h markedly augmented glucose-induced insulin secretion. This Wnt protein augmented glucose-induced insulin secretion was blocked by the addition of purified soluble Fizzled related protein 1 (sFRP1), a soluble form of Wnt receptors. In contrast, this augmentation of glucose-induced insulin secretion by Wnt-3a-CM was not seen in uninfected LRP5-/- islets. These data demonstrate that Wnt-3a-augmented glucose-induced insulin secretion is mediated by LRP5. In contrast to the augmentation by Wnt-3a of glucose-induced insulin secretion, the intracellular insulin levels were unchanged, indicating that Wnt-3a has no effects on the production of insulin in the islets.
Our data have demonstrated that LRP5 plays dual roles in the normal adult: facilitating the hepatic clearance of chylomicron remnants and stimulating glucose-induced insulin secretion from the islets. Impaired hepatic clearance of chylomicron remnants and glucose-induced insulin secretion lead to two disorders common in western countries: dietary-induced hypercholesterolemia and type 2-diabetes, respectively.

PUBLICATION

1. Sakakibara I, Fujino T, Ishii M, Tanaka T, Shimosawa T, Miura S, Zhang W, Tokutake Y, Yamamoto J, Awano M, Iwasaki S, Motoike T, Okamura M, et al. Fasting-induced hypothermia and reduced energy production in mice lacking acetyl-CoA synthetase 2 Cell Metab. 2009 Feb;9(2):191-202.
2. Iguchi H, Urashima Y, Inagaki Y, Ikeda Y, Okamura M, Tanaka T, Uchida A, Yamamoto TT, Kodama T, Sakai J. SOX6 suppresses cyclin D1 promoter activity by interacting with beta-catenin and histone deacetylase 1, and its down-regulation induces pancreatic beta-cell proliferation. J Biol Chem. 2007 Jun 29;282(26):19052-61. Epub 2007 Apr 4.
3. Gao J, Katagiri H, Ishigaki Y, Yamada T, Ogihara T, Imai J, Uno K, Hasegawa Y, Kanzaki M, Yamamoto TT, Ishibashi S, Oka Y. Involvement of apolipoprotein E in excess fat accumulation and insulin resistance. Diabetes. 2007 Jan;56(1):24-33.
4. Iguchi H, Ikeda Y, Okamura M, Tanaka T, Urashima Y, Ohguchi H, Takayasu S, Kojima N, Iwasaki S, Ohashi R, Jiang S, Hasegawa G, Ioka RX, et al. SOX6 attenuates glucose-stimulated insulin secretion by repressing PDX1 transcriptional activity and is down-regulated in hyperinsulinemic obese mice. J Biol Chem. 2005 Nov 11;280(45):37669-80. Epub 2005 Sep 7.
5. Ikeda Y, Iguchi H, Nakata M, Ioka RX, Tanaka T, Iwasaki S, Magoori K, Takayasu S, Yamamoto TT, Kodama T, Yada T, Sakurai T, Yanagisawa M, Sakai J. Identification of N-arachidonylglycine, U18666A, and 4-androstene-3,17-dione as novel insulin Secretagogues.
   Biochem Biophys Res Commun. 2005 Aug 5;333(3):778-86.
6. Iwasaki T, Takahashi S, Takahashi M, Zenimaru Y, Kujiraoka T, Ishihara M, Nagano M, Suzuki J, Miyamori I, Naiki H, Sakai J, Fujino T, Miller NE, Yamamoto TT, Hattori H. Deficiency of the very low-density lipoprotein (VLDL) receptors in streptozotocin-induced diabetic rats: insulin dependency of the VLDL receptor. Endocrinology. 2005 Aug;146(8):3286-94. Epub 2005 May 5.
7. Iwasaki T, Takahashi S, Ishihara M, Takahashi M, Ikeda U, Shimada K, Fujino T, Yamamoto TT, Hattori H, Emi M. The important role for betaVLDLs binding at the fourth cysteine of first ligand-binding domain in the low-density lipoprotein receptor. J Hum Genet. 2004;49(11):622-8.
8. Takahashi S, Sakai J, Fujino T, Hattori H, Zenimaru Y, Suzuki J, Miyamori I, Yamamoto TT
   The very low-density lipoprotein (VLDL) receptor: characterization and functions as a peripheral lipoprotein receptor. J Atheroscler Thromb. 2004;11(4):200-8.
9. Mashek DG, Bornfeldt KE, Coleman RA, Berger J, Bernlohr DA, Black P, DiRusso CC, Farber SA, Guo W, Hashimoto N, Khodiyar V, et al. Revised nomenclature for the mammalian long-chain acyl-CoA synthetase gene family. J Lipid Res. 2004 Oct;45(10):1958-61.
10. Yamamoto J, Ikeda Y, Iguchi H, Fujino T, Tanaka T, Asaba H, Iwasaki S, Ioka RX, Kaneko IW, Magoori K, Takahashi S, Mori T, Sakaue H, Kodama T, Yanagisawa M, Yamamoto TT, Ito S, Sakai J. A Kruppel-like factor KLF15 contributes fasting-induced transcriptional activation of mitochondrial acetyl-CoA synthetase gene AceCS2. J Biol Chem. 2004 Apr 3;279(17):16954-62.
11. Takahashi S, Sakai J, Fujino T, Miyamori I, Yamamoto TT. The very low density lipoprotein (VLDL) receptor--a peripheral lipoprotein receptor for remnant lipoproteins into fatty acid active tissues. Mol Cell Biochem. 2003 Jun;248(1-2):121-7.
12. Fujino T, Asaba H, Kang MJ, Ikeda Y, Sone H, Takada S, Kim DH, Ioka RX, Ono M, Tomoyori H, Okubo M, Murase T, Kamataki A, Yamamoto J, et al.Low-density lipoprotein receptor-related protein 5 (LRP5) is essential for normal cholesterol metabolism and glucose-induced insulin secretion. Proc Natl Acad Sci U S A. 2003 Jan 7;100(1):229-34.
13. Magoori K, Kang MJ, Ito MR, Kakuuchi H, Ioka RX, Kamataki A, Kim DH, Asaba H, Iwasaki S, Takei YA, Sasaki M, Usui S, Okazaki M, Takahashi S, Ono M, Nose M, Sakai J, Fujino T, Yamamoto TT. Severe hypercholesterolemia, impaired fat tolerance, and advanced atherosclerosis in mice lacking both low density lipoprotein receptor-related protein 5 and apolipoprotein E. J Biol Chem. 2003 Mar 28;278(13):11331-6.
14. Ioka RX, Kang MJ, Kamiyama S, Kim DH, Magoori K, Kamataki A, Ito Y, Takei YA, Sasaki M, Suzuki T, Sasano H, Takahashi S, Sakai J, Fujino T, Yamamoto TT. Expression cloning and characterization of a novel glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein, GPI-HBP1. J Biol Chem. 2003 Feb 28;278(9):7344-9..