α-, βII- and γ-subspecies of protein kinase C localized in the monkey hippocampus: pre- and post-synaptic localization of γ-subspecies

BRAIN RESEARCH ELSEVIER

Brain Research 656 (1994) 245 256

Research report

a-,/3 II- and ,/-subspecies of protein kinase C localized in the monkey hippocampus: pre- and post-synaptic localization of ,/-subspecies Naoaki Saito *, Takeshi Tsujino, Kaoru Fukuda, Chikako Tanaka Department of Pharmacology, Kobe University School oJ"Medicine, Kobe 650, Japan Accepted 31 May 1994

Abstract

Protein kinase C (PKC) has attracted wide attention as a key enzyme for the expression of long-term potentiation in the hippocampus, a basic model for memory. It is of interest to study the detailed localization of PKC subspecies in the monkey hippocampus. We used immunocytochemistry to examine the localization of PKC subspecies in the hippocampus of the monkey, Macaca mulatta. Subspecies of PKC in the monkey could be separated by hydroxyapatite chromatography and the elution profile proved to be similar to that of the rat. Antibodies against each a, /3II and y-subspecies of the rat specifically reacted with the respective subspecies of monkey PKC. The a-, /3II- and y-subspecies were distinctly distributed in the hippocampus. The /31-subspecies was not evident in the hippocampus. While both the a- and y-subspecies immunoreactive pyramidal cells were distributed throughout the hippocampus (CA1-CA3), the /3lI-subspecies immunoreactivc cells were scattered only in the CAI region. The y-subspecies was found in granule cells and dendrites in the dentate gyms, in mossy fibers and in their terminals in the CA3 region. The a-subspecies was also present in granule cells and in the dendrites but not in the mossy fibers. Glial cells did not stain with any of the antibodies used. Electron microscopy clearly showed that the y-subspecies was localized in both presynaptic terminals and post-synaptic dendrites. These observations suggest that subspecies of PKC in the monkey hippocampus may be involved in distinct functions and that the y-subspecies of PKC may act pre- and post-synaptically in pyramidal cells of the hippocampus. Key words: I m m u n o c y t o c h e m i s t r y ; E l e c t r o n microscopy; Monkey; Protein kinase C; h i p p o c a m p u s ; Subspecies: Presynapsc

1. Introduction

Protrin kinase C (PKC), a calcium-activated, phospholipid-dependent protein kinase, is abundant in the central nervous system and is involved in various neuronal signal transduction systems [31,32]. cDNAs encoding multiple subspecies of PKC (a, /3, 7, & E, r/, 0, A and () were isolated from various mammalian brain libraries: each subspecies had closely related but clearly different structures and was distinctly distributed in various tissues [7,22,33,34,36-40]. Enzymically, PKC of rat or rabbit could be resolved into at least three fractions [17,19] and each fraction corresponded to a,

* Corresponding author. Present address: Laboratory of Molecular Pharmacology, Biosignal Research Center, Kobe University, Rokkodai-cho 1-1, Nada-ku, Kobe 657, Japan. i Present address: Hyogo Institute for Aging Brain and Cognitive Disorders, 520 Saisho-Ko, Himeji 670, Japan.

0006-8993/94/$07.00 ~ 1994 Elsevier Science B.V. All rights reserved SSDI 0 0 0 6 - 8 9 9 3 ( 9 4 ) 0 0 6 7 2 - Y

/3, 7, 8, E and ~" subspecies [9,21]. Immunochemical and immunocytochemical studies also revealed the differential expression of each subspecies in the rat and rabbit brain [5,12,13,15,16,18,24,35,42,43,49]. The intracellular localization of each PKC subspecies also differed in various neurons in the rat brain; a - P K C was present in the periphery of the perikarya, /31-subspecies was just adjacent to the membrane, /31I-PKC was located around the Golgi complex, and y-PKC was homogeneously present in the cytoplasm [24,30,43,45, 48]. Long-term potentiation (LTP), which induces persistent enhancement of synaptic efficacy, has been considered a physiological model for learning and memory. LTP has been studied mainly in the hippocampal formation that contains the highest activity of PKC [44,47], and the involvement of PKC in LTP was deduced from electrophysiological results [4,8,14,27-29]. Immunocytochemical studies using subspecies-specific

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N. Saito eta/./Brain Research 656 (1994) 24.5 256

antibodies showed that c~-, /311- and y-PKCs were localized in the hippocampus of rat and that each subspecies had a different cellular and intracellular localization [18,23,41]. In primates, the hippocampus is also known to be involved in memory. As the enzymic properties and the precise localization of PKC subspecies in the monkey brain has been given less attention, we studied the chromatographic profile of monkey PKC. We describe here the cellular and intracellular localization of a-, /3II- and 7-PKCs in the monkey hippocampal formation.

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2. Experimental

2.1. Preparation of antibodies against PKC subspecies Rabbit antisera specific to the PKC subspecies were prepared against synthetic oligopeptides which corresponded to the carboxyl-terminal portion of each subspecies (QFVHPILQSAV for a-PKC, SYTNPEFVINV for /3I-PKC, and SFVNSEFLKPEVKS for /311PKC) [13,18,43]. A monoclonal antibody to y-PKC, obtained by immunizing rabbits with purified PKC from the soluble fraction of rat brain, was characterized [11].

2.2. Partial purification of PKC from monkey frontal cortex

B

a

80 kDa -

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III

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II

III

PKC activity was assayed by measuring the incorporation of 32p into bovine myelin basic protein (Sigma) from [T-32p]ATP in the presence of phosphatidylserine, diolein and Ca 2÷, as described [20]. PKC from monkey brain was partially purified from the soluble fraction of monkey hippocampus on a Mono Q column (5 × 5, Pharmacia H R 5 / 5 ) connected to an FPLC system (Pharmacia), then was resolved into three peaks, Type I, II, and III, by chromatography on a hydroxyapatite column, under the conditions described [21].

Fig. 1. Hydroxyapatite column chromatography of PKC from monkey hippocampus and immunoblot analysis. A: PKC activity was assayed under the conditions described in the text. e, in the presence of phosphatidylserine (16 /zg/ml), diolein (1.6 /xg/ml) and CaCI 2 (10 /zM).; o, in the presence of EGTA (0.5 raM) alone. Three peaks, Type I, II and III, used for the immunoblot (Fig. 1B) are indicated by arrows. Dashed line shows the concentration of potassium phosphate. B: immunoblot analysis of type I, II and III from monkey hippocampus. Three peaks of PKC activity was subjected to im~ munoblot analysis with the specific antibodies for a- (a),/311- (b) and y-PKC (c).

2.3. lmmunoblot analysis

2.4. Preparation of the tissue sections

The three peaks of PKC activity, Types I-III, were subjected to SDS-PAGE, followed by electrotransfer onto Immobilon (Millipore) membranes for Western blotting. The membrane were reacted with each antibody against a-, /3I-, /311- and y-PKC and stained using the peroxidase anti-peroxidase method, as described below.

The following steps were carried out at 4°C unless otherwise stated. Three rhesus monkeys (Macaca mulatta, Primate Research Institute Monkey Colony of Kyoto University) weighing 3.0-3.5 kg were deeply anesthetized by an intramuscular injection of ketamine (10 mg/kg) and intravenous administration of pentobarbital (50 mg/kg). The monkeys were perfused

Fig. 2. a-PKC immunoreactivity in the frontal section of the monkey hippocampus. A considerable number of pyramidal cells were stained in CAI-CA3, while there were no immunoreactive cells in the dentate gyrus (DG). Neuropil of the hippocampus showed homogeneous immunoreactivity except for the stratum lucidum in CA3. The neuropil was faintly stained in the proximal third of the molecular layer in the dentate gyrus. Square shows the subicular area where no pyramidal cells showed immunoreactivity. Bar = 500 ~m.

N. Saito et al. / Brain Research 656 (1994) 245-256

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through the left ventricle with 1 1 of saline, followed by 5 1 of fixative containing 4% paraformaldehyde (FA), 0.5% glutaraldehyde and 0.2% picric acid (PA) in 0.1

M phosphate buffer (PB, pH 7.4). After perfusion, the brain was removed, cut into blocks, immersed in a post-fixative solution containing 4% FA and 0.2% PA

I

Fig. 3./~II-PKC immunoreactivity in the frontal section of the monkey hippocampus. The/3II-PKC immunoreactive cells and neuropil was found only in the CAI region and not in CA3 and dentate gyrus (DG). Bar = 500/xm.

N. Saito et al. / Brain Research 656 (1994) 245-256

in 0.1 M PB for 48 h, and kept in 30% sucrose in 0.1 M phosphate buffer for several days. For light microscopic observations, the hippocampus was dissected, then frozen and sectioned frontally at 20 /xm thickness on a cryostat. The sections were dipped directly into 0.1 M phosphate-buffered saline (PBS, pH 7.4) containing 0.3% Triton-X and subsequently washed with the same buffer for at least 4 days at 4°C. For electron microscopy, the hippocampal blocks were

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frozen in liquid nitrogen, thawed, and cut into 40-/xm thick frontal sections on a vibratome.

2.5. Immunocytochemical procedures for the PKC subspecies The PBS used here contained 0.03% Triton-X (PBS-T) for light microscopic immunocytochemistry but not for electron microscopy. The sections were prein-

CA3

Fig. 4. The y-PKC immunoreactivity in the frontal section of the monkey hippocampus. The y-PKC immunoreactivity was distributed throughout the hippocampal formation. Dense immunoreactivity occurred in the fiber bundle-like structure in CA3. DG, dentate gyrus. Bar = 500/xm.

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N. Saito et al. / B r a i n Research 656 (1994) 245-250

N. Saito et al. /Brain Research 656 (1994) 245-256 cubated with 0.3% H 2 0 2 and 5% normal goat serum in PBS-T to block endogenous peroxidase activity and non-specific binding of the primary antibodies. The preparations were subsequently incubated with primary antibodies at appropriate concentrations in PBS-T containing 5% normal goat serum for 18 h at 4°C. After washing with PBS-T, the sections were incubated for an additional 4 h with goat anti-rabbit IgG ( G A R ) or goat anti-mouse IgG (GAM), then were incubated for 1.5 h with rabbit peroxidase anti-peroxidase complex (PAP) or mouse PAP complex. After three rinses, the preparations were developed with 0.02% 3,3'-diaminobenzidine, 0.2% nickel ammonium sulfate and 0.05% H202 in 50 mM Tris-HC1 (pH 7.4). The hippocampal sections were observed and photographed under a Zeiss light microscope. For electron microscopy, the immunostained sections were washed in PBS, post-fixed for 1 h in 2% osmium tetroxide in 0.1 M PBS, dehydrated in a graded series of ethanol and then flat-embedded on siliconized slides in Epon. After polymerization at 60°C for 48 h, the selected areas were cut off and attached to Epon supports for further sectioning on a Reichert-Jung Ultracut E ultramicrotome. Ultrathin sections were cut and mounted on 200 mesh uncoated grids ( M A X T A FORM), counterstained with 1% uranyl acetate in 50% ethanol, and examined under a JEM 100SX electron microscope.

3. Results

3.1. Hydroxyapatite chromatography of PKC and immunoblot analysis PKC activity of the monkey hippocampus was resolved into three peaks (Type I, II and III) on a hydroxyapatite column Fig. 1A. Immunoblot analysis showed that the antibodies against the a - P K C stained a single band of 80 kDa only in Type III, while antibodies against the /3II-PKC detected a 80 kDa band only in Type II (Fig. 1B). The y-PKC immunoreactive band was intense in Type I and a faint trace in Type II. /3I-PKC was not detected in any fraction. The immunoreaction was abolished by preincubation of the antibodies with the immunogen (data not shown).

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3.2. Immunocytochemical localization of PKC subspecies in the hippocampus 3. 2.1. Cellular distribution of PKC subspecies lmmunoreactivities of c~-, /3II- and y-PKC were evident in the monkey hippocampal formation whilst antibodies against /3I-PKC failed to stain. As shown in Figs. 2-4, the different distribution of each subspecies was evident in the hippocampus. The a - P K C immunoreactive cell bodies were abundant throughout C A 1 - C A 3 regions (Fig. 2) but were few in the subicular area. The strata radiatum and oriens were moderately stained in the C A I - C A 3 , although the stratum lucidum in the CA3 was immunonegative. The fiber bundle-like structure running parallel to the polymorphic layer was immunonegative among the faintly stained neuropil. In the dentate gyrus, the proximal third of the molecular layer had moderate a - P K C immunoreactivity. The /3II-PKC immunoreactivity was found only in the CA1 region of the hippocampus. The /3II-PKCpositive pyramidal cells were scattered in the CA1 and the neuropil of CA1 was also stained (Fig. 3). Littile of no immunoreactivity was seen in the CA2, CA3 region and dentate gyrus. The y - P K C immunoreactivity was distributed throughout the hippocampal formation. As shown in Fig. 4, a considerable number of immunoreactive pyramidal cells were present in the C A 1 - C A 3 . Neuropil of the hippocampus was moderately stained and the more intense immunoreactivity was found in the proximal portion of the stratum radiatum of the CA3 region. In the dentate gyrus, both the granule cell layer and the molecular layer were heavily stained. Furthermore, the y-PKC immunoreactive fiber bundle-like structure ran in parallel to the polymorphic layer. In the subicular area, a - P K C immunoreactivity was localized mainly in neuropil, while pyramidal cells did not stain (squared area in Figs. 2 and 5A). The immunonegative pyramidal cells in this area were surrounded by a-PKC-like immunoreactive dots (Fig. 5A, inset). In contrast, intense immunoreactivity was seen in the perikarya and the dendrites of most but not all pyramidal cells from the C A l - C A 2 region (Fig. 5B). Immunoreactive dendritic trees were seen among the neuropil which stained homogeneously. In the CA3

Fig. 5. a-PKC immunoreactivity in CA1, CA2, CA3 and dentate gyrus of the monkey hippocampal formation. A: subicular area. The a-PKC immunoreactivity was seen in the neuropil but not the pyramidal cells. Inset: the immunonegative pyramidal cell was surrounded by a- PKC immunoreaction. B: CA2. Pyramidal cells and their dendrites were intensely stained. The neuropil was also immunoreactive. Inset: dense immunoreactivity was found in the periphery of the perikaryon. C: CA3. Pyramidal cells and their dendrites were stained much as seen in CA2 but the neuropil in the stratum lucidum (asterisk) was not stained. Inset: immunoreactive dots were present on the plasma membrane. D: dot-like immunoreaction occurred in the granule cell layer and the immunoreactive dendrites were present in the inner third of the molecular layer. Arrowheads in insets show nucleoli. Bars = 50 txm for A; 100 ixm for B, C and D; 25 p~m for insets.

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region, the a-PKC immunoreactivity was similar to the distribution seen in the CA2, except that the neuropil did not stain in the stratum lucidum of the CA3 (Fig. 5C). The nuclei of the pyramidal cells did not stain in any region of the hippocampus. Under higher magnification, the immunoreactivity was located in the periphery of the perikarya and along the plasma membrane within the pyramidal cells of CA2 and CA3 (Fig. 5B,C, insets). In the dentate gyrus (Fig. 5D), dot-like immunoreaction was seen in the granule cell layer, and their dendrites were also immunoreactive, with more intense immunoreaction in the inner third of the molecular layer. The /3II-PKC immunoreactive cells were scattered in the CA1 region (Fig. 6A) and their dendrites were faintly stained. Intense dot-like immunoreaction was found mainly in the perikarya, and the dendritic networks were faintly stained (Fig. 6B). The /3II-PKC immunoreactivity was present in the perikarya but not on the plasma membrane (Fig. 6B, inset). Almost all pyramidal cells showed intense T-PKC immunoreactivity in the CA1 region of the hippocam-

pus (Fig. 7A). This intense immunoreactivity was found in the perikarya and there was moderate immunoreactivity in the nuclei of the pyramidal cells. The apical dendrites in the stratum radiatum and fine dendritic networks in the stratum lacunosum moleculare were evident among the y-PKC immunoreactive neuropil (Fig. 7A). In the CA3 region of the hippocampus, neuropil of the proximal portion of the stratum radiaturn showed a more intense immunoreactivity than seen in other layers (Fig. 7B). The intense immunoreactive fibers were visible from the stratum radiatum in CA3 region to the dentate gyrus passing through the polymorphic layer (Fig. 7B,C), thereby suggesting that the mossy fiber contained T-PKC. The perikarya and dendrites of the granule cells showed intense y-PKC immunoreactivity throughout the dentate gyrus (Fig. 7C).

3.2.2. Electron microscopic localization of y-PKC in CA1 and CA3 regions of the hippocampus When the monkey hippocampus was stained with each antibody for electron microscopic observation as

Fig. 6. /3II-PKC immunoreactivity in the CAI region of the monkey hippocampal formation. A: /~II-PKC immunoreactive cells were sparse. Bar = 100/.~m. B: intense dot-like immunoreaction was found mainly in the perikarya and dendrites. Bar = 50 ~zm. Insets: immunoreaction was present within the perikarya. Arrowhead points to nucleolus.

N. Saito et al. / B r a i n Research 656 (1994) 245-256

described above, adequate immunoreaction was obtained only with antibodies against 7-PKC, and the aand /3II-PKC immunoreactivities were too weak to be detected electron microscopically. In the CA1 region, y-PKC immunoreactivity was found in the presynaptic nerve terminals and also in the post-synaptic dendrites (Fig. 8A,B). The 7-PKC was present homogeneously in presynaptic terminals of the shaft synapse and appeared to be associated with synaptic vesicles (Fig. 8A). 7-PKC was also seen in some post-synaptic dendrites which made contact with non-immunoreactive synaptic terminals (Fig. 8B). The immunoreactivity was more intense with the synaptic density. Similarly, in the CA3 region, there were abundant immunoreactive presynaptic terminals contacting apical dendrites in the stratum lucidum (Fig. 8C).

4. Discussion

Monkey brain PKC was resolved into three fractions by hydroxyapatite chromatography. This has been done for the rat [17] and rabbit [19]. Three PKC isozymes (Type I, II and III) from rat or rabbit brain corresponded to y-, /3I + fill, and a-PKC, respectively [21]. Similarly, immunoblot analysis revealed that Type I, II and IIl from monkey brain corresponded to y-, /3- and

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a-subspecies of PKC, respectively. The faint y-PKC immunoreactivity in Type II is possibly caused by a trace of Type I. All three types of PKC had the same apparent molecular mass of 80 kDa. The antibodies raised against rat PKC subspecies specifically recognized each corresponding subspecies of monkey PKC. This suggests that the monkey PKC subspecies have antigenicity similar to that of rat PKC subspecies, and that these antibodies are useful to demonstrate localization of PKC subspecies in monkeys as well as in rats. PKC has attracted wide attention as an important factor for the maintenance of LTP [8,14,27-29], but the subspecies of PKC which contributes to LTP has remained unknown. Whether pre- or post-synaptic contribution of PKC is the more important for the maintenance of LTP is also unknown. It is of significance to study the detailed distribution of PKC in the monkey hippocampus. The post-synaptic contribution of PKC to LTP was mainly demonstrated in the CAI region of the hippocampus, an area where activation of the NMDA receptor is essential for the induction of LTP [6]. The persistent activity of PKC in post-synaptic cells is considered to be necessary for the maintenance of LTP [29]. The post-synaptic injection of PKC into pyramidal cells of CA1 elicited the LTP-like phenomenon [14], and the post-synaptic delivery of PKC inhibitors inhibited both the initial induction and the maintenance of

Fig. 7. The y-PKC immunoreactivity in CA1, CA3 and dentate gyrus of the monkey hippocampal formation. A: CA1. Most pyramidal cells and their dendritic trees were stained. Intense immunoreactivity was present in the perikarya and the nuclei were lightly stained. B: CA3. Neuropil of the proximal portion of the stratum radiatum (asterisk) in CA3 showed more intense immunoreactivity. The mossy fiber (arrowheads) was also immunoreactive. C: dentate gyrus. T h e perikarya and dendrites of the granule cells were intensely stained. An immunoreactive mossy fiber (arrowheads) was visible. Bars ~ 100/zm.

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LTP in CA1 [46]. Recent molecular cloning revealed the existence of multiple N M D A receptor subunits and the heteromeric N M D A channels expressed in X e t t o p u s oocytes were activated by treatment with phorbol ester [25]. Furthermore, the activation of PKC facilitated the N M D A response in CA1 hippocampal neurons [3]. These findings suggest that PKC may phosphorylate the N M D A receptor and facilitate N M D A currents in the initial stage of LTP. The present electron microscopic study showed that y - P K C was present in dendritic spines, especially on the post-synaptic density of excitatory synapses of CA1; thus, y - P K C may modulate the N M D A channels post-synaptically in the CA1 region of the monkey hippocampus. The presynaptic contribution of PKC to L T P was suggested by findings that glutamate release from presynaptic terminals is enhanced by phorbol esters in rat hippocampal slices [27], and that neuromodulin (protein F1, B-50, GAP-43), a presynaptic PKC substrate [10], was specifically phosphorylated by PKC during LTP [2,26]. In the rat hippocampus, a-, /311and y-PKCs were found in the post-synaptic but not in the presynaptic component [23] and e-PKC was predominantly localized in presynaptic terminals [41]. Therefore, mainly e-PKC rather than a-, /311- or yPKC, is probably involved in the presynaptic events of LTP such as the enhancement of glutamate release or the phosphorylation of neuromodulin. Interestingly, in the monkey hippocampus, y-PKC was localized both in presynaptic terminals and post-synaptic dendrites. It is possible that the antibody against y - P K C cross-reacts with monkey e-PKC that is localized in the synapttc terminals. However, from the immunoblot analysis, the e-PKC of 90 kDa was not recognized by the antibody against y-PKC, therefore, the y - P K C like immunoreactivity in the presynaptic terminals indicates the existence y-PKC but not e-PKC, y - P K C may be involved in presynaptic functions in the monkey hippocampus. The distribution in the monkey hippocampus, however. might not be directly of significance to the mechanism of rat LTP. A different intracellular localization of y-subspecies between rat and monkey may be due to the quantity of this enzyme in the presynaptic terminals, but the abundant presynaptic localization of yPKC suggests that this particular subspecies of PKC

Fig. 8. Electron micrographs showing y-PKC immunoreactivity in CA1 and CA3 regions of the hippocampus. A,B: CA1. The y-PKC immunoreactivity was found in the presynaptic nerve terminals (A) and the immunoreactive terminals made synaptic contacts with immunonegative dendrites. The immunoreactivitywas homogeneous in synaptic terminals, y-PKC was also seen in the post-synaptic dendrites (B). The immunoreactive dendrites received non-immunoreactive synaptic terminals. Arrows show post-synaptic densities. C,D: CA3. The immunoreactive terminals (C) were also seen in CA3. Arrows show post-synaptic densities. Bars = 0.5 p,m.

N. Saito et al. / Brain Research 656 (1994) 245-256

may play a special role in presynaptic plasticity in primates. A recent study revealed that LTP was greatly diminished in mice deficient in y-PKC [1]. The functional role of T-PKC in the expression of LTP appeared to be of great interest in the study of learning and memory. In addition, as a-PKC immunoreactivity was seen to surround pyramidal cells in the subicular area, a-PKC may also be associated with presynaptic events. In the CA3 region there was a unique localization of monkey PKC subspecies. The proximal portion of the stratum radiatum where the mossy fibers terminate was densely labeled for y-PKC but was devoid of staining for ce-PKC, while the post-synaptic dendrites of the pyramidal cells were stained for both a- and y-PKC. This suggests that y-PKC but not c~-PKC is involved in the presynaptic modulation of mossy fiber-CA3 synapses and that both of the subspecies are involved in the 13ost-synaptic modulation in monkey hippocampus.

Acknowledgements This work was supported by research grants from the Scientific Research Fund of the Ministry of Education, Science, and Culture, Japan, from the Yamanouchi Foundation for Research on Metabolic Disorders, from the Osaka Cancer Research Fund, from Uehara Memorial Foundation and from Yokoyama Foundation for Clinical Pharmacology. We thank K. Kubota and T. Oishi for their critical advice and thank M. Ohara for reading the manuscript.

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