Introduction

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Physiological hyperketonemia occurs quite readily via "Randle's glucose-fatty acid cycle" as a compensatory defensive response against fasting, particularly in the neonate and pregnancy, occasionally developing into frank ketoacidosis (Féry and Balasse, 1985; Laffel, 1999). Also, hyperketonemia is brought about during prolonged exercise and high-fat diet in normal individuals. In patients with congenital enzyme defects unable to catalyze hepatic mitochondrial synthesis of ketone bodies, even short-term fasting causes hypoketotic hypoglycemia, increased levels of plasma free fatty acids (FFA), and childhood sudden death because of their inability to oxidize FFA into ketone bodies (Hashimoto et al., 2000). In contrast, diabetic ketoacidosis due to the defective insulin secretion and insulin resistance is a life-threatening state. In patients with diabetes mellitus, obesity, and atherosclerotic vascular diseases, the increased levels of plasma FFA are linked to the insulin-resistant state in these diseases (Boden et al., 2001) because FFA interfere with insulin's intracellular signaling (Patti, 1999). Density of cell surface insulin receptors (IR), a member of the receptor tyrosine kinase family, is a key determinant in regulating the strength of insulin's acute and chronic pleiotropic effects, such as the metabolic and neurotropic effects (Dikic et al., 1994). In pancreatic -cells, evidence has emerged that IR regulate secretion (Aspinwall et al., 2000) and synthesis (Leibiger et al., 1998; Jackerott et al., 2001) of insulin via an autocrine manner. Neonatal mouse lacking IR rapidly developed into the lethal diabetic ketoacidosis (Jackerott et al., 2001).

IR consist of two extracellular -subunits (~135 kDa) and two transmembrane -subunits (~95 kDa), which are encoded by the same gene and derived from the single chain IR precursor molecule (~190 kDa) (Cheatham and Kahn, 1995). IR precursor undergoes cotranslational glycosylation, intrachain disulfide-bond formation/isomerization, and disulfide-linked homodimerization at the endoplasmic reticulum (ER). The homodimeric IR precursor is proteolytically processed into the disulfide-linked ₂₂ complex at the trans-Golgi network, which is transported to the plasma membrane via an as yet unidentified mechanism (Cheatham and Kahn, 1995). Binding of insulin to the -subunit causes autophosphorylation of the -subunit, leading to the endocytic internalization of IR via clathrin-coated vesicles. IR internalization may trigger phosphorylation of insulin receptor substrate-1 (IRS-1) at the multiple tyrosine residues, which serve as binding sites for various signaling molecules containing the Src homology-2 domain, thus triggering insulin's pleiotropic effects (Cheatham and Kahn, 1995).

In adrenal chromaffin cells (embryologically derived from the neural crest), IR play pivotal roles, such as up-regulation of cell surface voltage-dependent Na⁺ channels (Yamamoto et al., 1996) and enhancement of voltage-dependent Ca²⁺ channel gating and of exocytic secretion of catecholamines (Yamamoto et al., 1996), as well as increased synthesis of various bioactive peptides (e.g., enkephalin) contained within chromaffin granules (Wilson et al., 1985). Our previous study showed that chaperone function of the heat shock protein 90-kDa family was indispensable to the homodimerization of monomeric IR precursor at the ER (Saitoh et al., 2002). Protein kinase C- increased IR mRNA and protein levels, thus causing up-regulation of cell surface IR (Yamamoto et al., 2000). Also, peptidyl prolyl cis-trans-isomerase activity of immunophilins (Shiraishi et al., 2000) and Ca²⁺-ATPase activity of the ER (Shiraishi et al., 2001) promoted cell surface externalization of IR from the trans-Golgi network. In the present study, ¹²⁵I-insulin binding, immunoblot, and Northern blot analyses show that chronic treatment with acetoacetate (but not -hydroxybutyrate, acetone, and acidic medium) selectively down-regulated cell surface expression of IR via lowering IR mRNA levels and IR synthesis, thereby attenuating insulin-induced tyrosine-phosphorylation of IRS-1.

	Materials and Methods

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Materials. Eagle's minimum essential medium was from Nissui Seiyaku (Tokyo, Japan). Calf serum, phenylmethylsulfonyl fluoride, leupeptin, aprotinin, sodium orthovanadate, sodium fluoride, Nonidet P-40, Tween 20, and NaN₃ were from Nacalai Tesque (Kyoto, Japan). Acetoacetate, dl--hydroxybutyrate, clofibrate, 6-carba-prostaglandin I₂ (cPGI₂), oleic acid, arachidonic acid, eicosapentaenoic acid, brefeldin A, and actinomycin D were from Sigma-Aldrich (St. Louis, MO). Acetone was from Wako Pure Chemicals (Osaka, Japan). Troglitazone was kindly donated from Sankyo (Tokyo, Japan). Rabbit polyclonal antibody against the human IR -subunit was from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit polyclonal IRS-1 antibody was from Upstate Biotechnology (Lake Placid, NY). Mouse monoclonal phosphotyrosine-specific antibody (PY-20) was from BD PharMingen (San Diego, CA). Protein A-agarose and Oligotex-dT30<Super> were from Nippon Roche (Tokyo, Japan). TRIzol reagent was from Invitrogen (Carlsbad, CA). The BcaBEST labeling kit and Noninterfering Protein Assay kit were from Takara (Kyoto, Japan). ¹²⁵I-Labeled donkey anti-rabbit IgG, ¹²⁵I-insulin (~2000 Ci/mmol), and [-³²P]dCTP (>4000 Ci/mmol) were from Amersham Biosciences (Piscataway, NJ). ¹²⁵I-Insulin was diluted with nonradioactive human insulin Humulin R (Eli Lilly, Kobe, Japan), and ¹²⁵I-insulin (3.125 Ci/mmol) was used for the ¹²⁵I-insulin binding assay. cDNA for human glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) was from CLONTECH Laboratories (Palo Alto, CA). Plasmid containing human IR cDNA (pSELECT HIR) was generously donated from Drs. Graeme Bell and Donald F. Steiner (Yamamoto et al., 2000).

Primary Culture of Adrenal Chromaffin Cells: Treatment with Test Compounds and Acidic Medium. Isolated bovine adrenal chromaffin cells were cultured (4 × 10⁶ per dish, Falcon; 35-mm diameter) in Eagle's minimum essential medium containing 10% calf serum under 5% CO₂/95% air in a CO₂ incubator (Yamamoto et al., 1996, 2000). Three days (60-62 h) later, the cells were treated in the fresh culture medium with or without acetoacetate, -hydroxybutyrate, acetone, clofibrate, cPGI₂, or troglitazone for up to 48 h or exposed to freshly prepared acidic culture medium (pH 6.9) for 24 h. The pH values of these test compound-containing media were 7.4, as measured by pH meter F-14 (Horiba, Kyoto, Japan). The acidic medium was prepared by adding 1 N HCl to the culture medium, its pH value being reconfirmed as 6.9 after the 24-h incubation period. Ketone bodies and other test compounds were dissolved in dimethyl sulfoxide (DMSO) and ethanol, respectively. The final concentrations (~0.2%) of DMSO and ethanol in the test medium did not affect ¹²⁵I-insulin binding capacity, IR precursor, and IR -subunit levels, or the basal tyrosine-phosphorylation level of IRS-1. The culture medium contained 3 µM cytosine arabinoside to suppress the proliferation of nonchromaffin cells; when chromaffin cells were further purified by differential plating (Yamamoto et al., 1996, 2000), ¹²⁵I-insulin binding was similar between purified and conventional chromaffin cells. Also, acetoacetate treatment (10 mM for 24 h) decreased ¹²⁵I-insulin binding by 27 and 28% in purified and conventional chromaffin cells compared with nontreated cells in each cell group.

¹²⁵I-Insulin Binding. Cells were washed with ice-cold Krebs-Ringer phosphate (KRP) buffer (154 mM NaCl, 5.6 mM KCl, 1.1 mM MgSO₄, 2.2 mM CaCl₂, 0.85 mM NaH₂PO₄, 2.15 mM Na₂HPO₄, 5 mM glucose, and 0.5% bovine serum albumin, pH 7.4) and incubated with 0.025 to 10 nM ¹²⁵I-insulin in 1 ml of KRP buffer at 4°C for 6 h in the absence (total binding) and presence (nonspecific binding) of 1 µM unlabeled insulin. The cells were immediately washed, solubilized in 0.2 M NaOH, and counted for radioactivity. Specific binding was calculated as the total binding minus nonspecific binding. The B_max and K_d values of ¹²⁵I-insulin binding correspond to the binding of ¹²⁵I-insulin to IR (but not insulin-like growth factor receptors), as reported previously (Yamamoto et al., 1996, 2000; Shiraishi et al., 2000, 2001). ¹²⁵I-Insulin binding represents cell surface (but not internalized) IR because ¹²⁵I-insulin associated with chromaffin cells were completely removed by washing the cells with ice-cold acidic (pH 4.0) KRP buffer twice, each for 7 min, as reported previously (Yamamoto et al., 2000).

Western Blot Analysis of IR and the IR Precursor Molecule. Cells were washed with ice-cold Ca²⁺-free phosphate-buffered saline and solubilized in 500 µl of 2× SDS electrophoresis sample buffer [125 mM Tris-HCl (pH 6.8), 20% glycerol, 10% 2-mercaptoethanol, and 4% SDS] at 98°C for 3 min. Total quantities of cellular proteins, as measured by the Noninterfering Protein Assay kit, were not changed between nontreated and ketone body-treated or acidic medium-treated cells. The same amounts of proteins (7.0-7.5 µg/lane) were separated by SDS-7.5% polyacrylamide gel electrophoresis (PAGE) and transferred onto a nitrocellulose membrane; it was preincubated with 5% dry milk in phosphate-buffered saline and reacted overnight at 4°C with rabbit antibody against the C-terminal amino acid sequence (1365-1382) of the IR -subunit (Cheatham and Kahn, 1995; Shiraishi et al., 2000, 2001; Yamamoto et al., 2000; Saitoh et al., 2002). After repeated washings, the immunoreactive bands were labeled with ¹²⁵I-anti-rabbit IgG (1:1000) and analyzed by a Bioimage BAS 2000 analyzer (Fuji Film, Tokyo, Japan).

Northern Blot Analysis of IR mRNA Levels. Total cellular RNA was isolated from cells by acid guanidine-thiocyanate-phenol-chloroform extraction using TRIzol reagent. poly(A)⁺RNA was purified by Oligotex-dT30<Super>, electrophoresed on 1% agarose gel containing 6.3% formaldehyde in buffer [40 mM 3-(N-morpholino) propanesulfonic acid (pH 7.2), 0.5 mM EDTA, and 5 mM sodium citrate], transferred to a nylon membrane (Hybond-N; Amersham Biosciences) in 20× saline-sodium citrate (SSC; 1× SSC = 0.15 M NaCl and 0.015 M sodium citrate) overnight and cross-linked using a UV cross-linker (Funakoshi, Tokyo, Japan). The IR cDNA fragment (nucleotides 1-4608), obtained by digestion of pSELECT HIR with SalI, and GAPDH cDNA (1.1 kilobase pairs) were labeled with [-³²P]dCTP using the BcaBEST labeling kit (Yamamoto et al., 2000; Saitoh et al., 2002). The membrane was prehybridized at 42°C in 6× SSC, 10× Denhardt's solution (2% bovine serum albumin fraction V, 2% polyvinylpyrrolidone, and 2% Ficoll 400), 50% formamide, 0.5% SDS, and 50 µg/ml salmon sperm DNA and then hybridized with the IR probe under the same condition for 18 h. It was washed at 55°C in 2×, 1×, and 0.2× SSC containing 0.1% SDS, each for 30 min twice, and subjected to autoradiography. The same membrane was hybridized with the GAPDH probe after it was thoroughly washed in 0.1% SDS at 100°C to remove the IR probe. The autoradiogram was quantified by a Bioimage BAS 2000 analyzer.

Immunoprecipitation, PAGE, and Immunoblot Analysis of IRS-1 and Tyrosine-Phosphorylated IRS-1. Cells were washed, solubilized at 4°C for 15 min in 1 ml of lysis buffer [50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 1 mM phenylmethylsulfonyl fluoride, 10 mM EDTA, 20 µg/ml aprotinin, and 10 µg/ml leupeptin], and centrifuged at 12,000g for 10 min at 4°C. The supernatant was reacted with protein A-agarose for 1 h at 4°C and centrifuged. Proteins in the supernatant were immunoprecipitated with IRS-1 antibody for 2 h at 4°C and then with protein A-agarose for 1 h. The immunoprecipitate was washed three times with the lysis buffer by repeated resuspension and centrifugation, solubilized in 25 µl of 2× SDS electrophoresis sample buffer at 98°C, and centrifuged to remove protein A-agarose. Proteins in the supernatant were size-fractionated by SDS-PAGE and transferred to membrane for immunoblot analysis of IRS-1 level. To measure insulin-induced tyrosine-phosphorylation of IRS-1, cells were washed with KRP buffer and incubated at 37°C with or without 100 nM insulin for 10 min in 1 ml of KRP buffer; cells were solubilized in the lysis buffer containing 100 mM NaF and 10 mM Na₃VO₄ and subjected to immunoprecipitation with IRS-1 antibody.

Statistical Methods. ¹²⁵I-Insulin binding was performed in triplicate, and all experiments were repeated at least three times (mean ± S.E.M.). Significance (p < 0.05) was determined by one-way or two-way analysis of variance with post hoc mean comparison by Newman-Keuls multiple range test. Student's t test was used when two means of a group were compared.

	Results

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Reduction of Cell Surface ¹²⁵I-Insulin Binding by Chronic Treatment with Acetoacetate: No Effect of -Hydroxybutyrate, Acetone, Acidic Medium, Clofibrate, cPGI₂, and Troglitazone. It has been shown that in patients with noninsulin-dependent diabetes mellitus under good nutritional status, plasma concentrations of acetoacetate and -hydroxybutyrate ranged between 0.25 to 3.05 and 0.66 to 10.23 mM, respectively (Féry and Balasse, 1985). Total plasma ketone bodies (acetoacetate plus -hydroxybutyrate) are >1 mM in hyperketonemia and >3 mM in ketoacidosis, developing to >25 mM with the preferential increase of -hydroxybutyrate in diabetic ketoacidosis (Laffel, 1999). As shown in Fig. 1A, treatment of adrenal chromaffin cells with 10 mM acetoacetate for 24 h decreased cell surface ¹²⁵I-insulin binding capacity by 28%, whereas the same treatment with 10 mM -hydroxybutyrate, 100 mM acetone, or acidic medium (pH 6.9) had no effect. Figure 1B shows that the decreasing effect of acetoacetate on ¹²⁵I-insulin binding became evident between 12 and 24 h, the reduction attaining to 28, 36, and 38% at 24, 36, and 48 h, respectively. Cells were treated with acetoacetate for the first 24 h, then washed (Fig. 1B, arrow), and incubated in the absence of acetoacetate for up to 48 h; ¹²⁵I-insulin binding gradually returned to the control nontreated level between 24 and 48 h. Figure 1C shows that acetoacetate decreased ¹²⁵I-insulin binding in a concentration-dependent manner between 3 and 60 mM. Rosenthal plot analysis (Fig. 1D) revealed that acetoacetate (10 mM for 24 h) decreased the B_max values, with a statistical significance (P < 0.05) from 115.5 ± 4.2 to 83.2 ± 3.8 fmol/4 × 10⁶ cells, without altering the K_d values (3.5 ± 0.3 nM, nontreated cells; 3.4 ± 0.3 nM, acetoacetate-treated cells; n = 5).

View larger version (35K): [in this window] [in a new window]   Fig. 1.   Chronic treatment of adrenal chromaffin cells with acetoacetate, -hydroxybutyrate, acetone, and acidic medium: their effects on cell surface 125I-insulin binding. A, cells were treated without (vehicle) or with 10 mM acetoacetate, 10 mM -hydroxybutyrate, 100 mM acetone or acidic medium (pH 6.9) for 24 h, and 125I-insulin binding was assayed. B, cells were treated with () or without () 10 mM acetoacetate for up to 48 h and subjected to 125I-insulin binding assay at the indicated times. In parallel study, cells were treated with 10 mM acetoacetate for the first 24 h, washed (indicated by arrow), then incubated in the absence () of acetoacetate, and subjected to 125I-insulin binding assay at 36 and 48 h. C, cells were treated with () or without () 0.3 to 60 mM acetoacetate for 24 h and subjected to 125I-insulin binding assay. Mean ± S.E.M. (n = 3). D, Rosenthal plot of 125I-insulin binding to the cells treated with () or without () 10 mM acetoacetate for 24 h. The plot is typical from five separate experiments with similar results (inset). *, P < 0.05 compared with nontreated cells; #, P < 0.05 compared with acetoacetate-treated cells; B/F, bound/free.

Internalization Rate of Cell Surface IR: No Effect of Chronic Treatment with Acetoacetate. By using brefeldin A, we examined whether acetoacetate may accelerate the internalization rate of cell surface IR. Brefeldin A is an inhibitor of guanine nucleotide-exchange protein of ADP-ribosylation factor 1, a monomeric GTPase. Previous studies showed that brefeldin A treatment (2.5-10 µg/ml for 2-36 h) of various intact cells blocks cell surface externalization from the trans-Golgi network of newly synthesized transferrin receptors, _1B-adrenoceptors, renal epithelial sodium channels, and glucose transporter-4, whereas having no effect on ADP-ribosylation factor 6-catalyzed internalization of cell surface receptors and ion channels (Shiraishi et al., 2000, 2001; Yamamoto et al., 2000). In adrenal chromaffin cells, previous fluorescence study showed that brefeldin A treatment (0.28-2.8 µg/ml for 2 h) was sufficient to cause disassembly of the Golgi membrane in most (>90%) chromaffin cells (Xu and Tse, 1999).

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Internalization of cell surface IR: no effect of acetoacetate treatment. In the absence () or presence (, ) of 10 µg/ml brefeldin A, cells were treated with () or without () 10 mM acetoacetate for up to 18 h; at each indicated incubation time, the remaining cell surface IR was measured by 125I-insulin binding assay. Mean ± S.E.M. (n = 5). *, P < 0.05 compared with brefeldin A-nontreated cells.

Cellular Levels of Immunoreactive IR -Subunit and IR Precursor Molecule: Reduction by Chronic Treatment with Acetoacetate, but Not with -Hydroxybutyrate, Acetone, and Acidic Medium. Western blot analysis (Fig. 3A) shows that the IR -subunit antibody recognized single major (~95 kDa) and single minor (~190 kDa) bands, which is in agreement with the molecular sizes of the mature IR -subunit and IR precursor molecule, respectively (Cheatham and Kahn, 1995; Shiraishi et al., 2000, 2001; Yamamoto et al., 2000; Saitoh et al., 2002). The antibody, when reacted with its immunogen before the immunoblot analysis, did not recognize these bands (data not shown). Quantification of these immunoreactive bands (Fig. 3B) shows that acetoacetate (10 mM for 24 h) lowered cellular levels of the IR -subunit and IR precursor molecule by 28 and 22%, respectively. In contrast, the same 24-h treatment with 10 mM -hydroxybutyrate, 100 mM acetone, or acidic medium (pH 6.9) had no effect on the IR -subunit and IR precursor levels.

View larger version (37K): [in this window] [in a new window]   Fig. 3.   Western blot analysis of the IR -subunit and IR precursor levels: reduction by chronic treatment with acetoacetate, but not with -hydroxybutyrate, acetone, and acidic medium. A, cells were treated without (none) or with DMSO (vehicle), 10 mM acetoacetate, 10 mM -hydroxybutyrate, 100 mM acetone or acidic medium (pH 6.9) for 24 h and solubilized in SDS electrophoresis sample buffer; proteins were separated by SDS-PAGE under the reducing condition, transferred to membrane, and then subjected to immunoblot analysis using antibody raised against IR -subunit. Blot shown is typical from three independent experiments with similar results. B, levels of IR -subunit and IR precursor in A were quantified by a Bioimage analyzer; the level obtained in nontreated cells (none) is assigned a value of 100%. *, P < 0.05 compared with DMSO-treated cells.

IR mRNA Levels: Reduction by Chronic Treatment with Acetoacetate. As shown in Fig. 4A, the IR cDNA probe hybridized to one major (~9.4 kb) and two minor (~7.0 and ~5.0 kb) IR transcripts, consistent with the molecular sizes of multiple species of IR mRNAs; these multiple transcripts encompass, in addition to the coding regions, different lengths of 5'- and 3'-untranslated regions (Yamamoto et al., 2000; Saitoh et al., 2002). IR mRNA levels were normalized against GAPDH mRNA levels (Fig. 4B). Levels of 9.4-, 7.0-, and 5.0-kb IR mRNAs were decreased, respectively, by 23, 19, and 20% as soon as 6 h after acetoacetate (10 mM) treatment and remained decreased at 12 and 24 h.

View larger version (33K): [in this window] [in a new window]   Fig. 4.   Northern blot analysis of IR mRNA levels: reduction by chronic treatment with acetoacetate. A, cells were treated with (+) or without () 10 mM acetoacetate for up to 24 h; poly(A)+RNA was extracted, electrophoresed on agarose gel, and transferred to membrane. The membrane was sequentially hybridized to each 32P-labeled cDNA probe for IR and GAPDH after removing the former probe. Data are typical from three separate experiments with similar results. B, levels of IR mRNAs and GAPDH mRNA in A were quantified by a Bioimage analyzer, and the relative amounts of IR mRNAs [9.4 kb (), 7.0 kb (), and 5.0 kb ()]/GAPDH mRNA were calculated. A value of 100% represents the relative level obtained in nontreated cells at the left lane in each incubation time. C, cells were pretreated for 6 h with or without 10 mM acetoacetate (Fig. 4B) and incubated with 10 µg/ml actinomycin D in the continuous absence or presence of acetoacetate. At the indicated times, poly(A)+RNA was isolated and subjected to Northern blot analysis. The level of IR major mRNA (~9.4 kb) was quantified by a Bioimage analyzer. Mean ± S.E.M. (n = 3). *, P < 0.05 compared with acetoacetate-nontreated cells.

Attenuation of Insulin-Induced Tyrosine-Phosphorylation of IRS-1 in Acetoacetate-Treated Cells: No Effect on IRS-1 Level. Because IRS-1 mediates most, if not all, effects of IR (Cheatham and Kahn, 1995; Aspinwall et al., 2000), we examined whether acetoacetate-induced down-regulation of cell surface IR may decrease the intrinsic tyrosine kinase activity of IR, thereby affecting insulin-induced tyrosine-phosphorylation of IRS-1. In nontreated and acetoacetate-treated (10 mM for 24 h) cells (Fig. 5A), the cell lysates were immunoprecipitated with IRS-1 antibody, followed by SDS-PAGE, and then subjected to immunoblot analysis with IRS-1 antibody; cellular levels of IRS-1 were comparable between nontreated and acetoacetate-treated cells (upper panel, lanes 1 to 4). In contrast, when nontreated and acetoacetate-treated cells were incubated with or without 100 nM insulin for 10 min (lower panel, lanes 1 to 4), insulin-induced tyrosine-phosphorylation of IRS-1 was attenuated by 56% in acetoacetate-treated cells compared with nontreated cells (Fig. 5B).

View larger version (32K): [in this window] [in a new window]   Fig. 5.   Reduction of insulin-induced tyrosine-phosphorylation of IRS-1 in acetoacetate-treated cells. A, cells were treated with (+) or without () 10 mM acetoacetate for 24 h and incubated with (+) or without () 100 nM insulin for 10 min. Cell lysates were subjected to immunoprecipitation (IP) with IRS-1 antibody; the immunoprecipitates were separated by SDS-PAGE, transferred to membrane, and subjected to immunoblot (IB) analysis using IRS-1 antibody (upper panel) or phosphotyrosine-specific antibody (anti-pTyr) (lower panel). Data are typical from three independent experiments with similar results. IRS-1-P, tyrosine-phosphorylated form of IRS-1. B, levels of insulin-induced tyrosine-phosphorylation of IRS-1 in A were quantified by a Bioimage analyzer; a value of 100% represents the basal phosphorylation in cells that were incubated at 37°C for 24 h in the absence of acetoacetate and DMSO. Mean ± S.E.M. (n = 3). *, P < 0.05 compared with nontreated cells.

	Discussion

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Down-Regulation of Cell Surface IR Caused by Chronic Treatment with Acetoacetate. Our present study showed that chronic (24 h) treatment with acetoacetate (3 mM) decreased ¹²⁵I-insulin binding capacity and cellular levels of the IR precursor molecule and mature IR -subunit, which were attributed to the retardation of IR synthesis. Northern blot analysis documented that acetoacetate treatment lowered IR mRNA levels by ~23% as early as 6 h when cell surface ¹²⁵I-insulin binding capacity was not yet decreased in acetoacetate-treated cells. The decrease of IR mRNA levels further developed at 12 and 24 h, and reduction of cell surface ¹²⁵I-insulin binding became evident between 12 and 24 h. In addition, acetoacetate shortened t_1/2 of IR mRNA from 13.6 to 9.5 h. Thus, acetoacetate may retard IR synthesis by lowering steady-state levels of IR mRNAs, which is attributed, at least in part, to the decreased stability of IR mRNA in acetoacetate-treated cells. mRNA stability is regulated by the specific interaction between nucleotide cis-elements and trans-acting nucleotide regulatory proteins; the trans-acting proteins are constitutively expressed or stimuli-inducible and shuttle between nucleus and cytoplasm (Shyu and Wilkinson, 2000; Guhaniyogi and Brewer, 2001). Nucleotide cis-elements and trans-acting nucleotide regulatory proteins that regulate transcription of the IR gene have been increasingly identified (Yoshizato et al., 2001). Much, however, remains unknown about the extra- and intracellular signaling molecules that regulate IR mRNA stability. Our present study may provide the first evidence that chronic treatment with acetoacetate down-regulates IR mRNA levels by destabilizing IR mRNA, thus lowering synthesis and cell surface expression of IR. In addition, the reduction of ¹²⁵I-insulin binding caused by acetoacetate was reversible as early as 12 h after the washout of acetoacetate-treated (10 mM for 24 h) cells. This observation raises the possibility that acetoacetate may regulate the stability of IR mRNA in a moment-to-moment manner.

Biological Relevance of Acetoacetate-Induced Down-Regulation of Cell Surface Functional IR. In acetoacetate-treated (10 mM for 24 h) cells, our present study showed that insulin-induced tyrosine-phosphorylation of IRS-1 was attenuated by 56%, with no change in the level of IRS-1. Because the K_d values of cell surface ¹²⁵I-insulin binding were similar between nontreated and acetoacetate-treated cells, the attenuation of insulin-induced tyrosine-phosphorylation of IRS-1 in acetoacetate-treated cells is attributed to the acetoacetate-induced down-regulation of cell surface functional IR. In contrast to our present study, Dikic et al. (1994) documented that increased expression of IR in PC12 cells caused enhancement of insulin-induced activation of IRS-1, Sos, and Shc, as well as nuclear translocation of mitogen-activated protein kinase that normally remained in the cytoplasm following insulin stimulation in the native PC12 cells. Also, the increased expression of IR in PC12 cells converted insulin's biological effect from cell proliferation to cell differentiation into the neuronal cells (Dikic et al., 1994). Thus, acetoacetate-induced aberrant down-regulation of cell surface IR and the subsequent attenuated tyrosine-phosphorylation of IRS-1 may perturb insulin's biological effects in a quantitative and qualitative manner.