Cooling-increased phospho-β-arrestin-1 and β-arrestin-1 expression levels in 3T3-L1 adipocytes

Cryobiology 65 (2012) 12–20

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Cooling-increased phospho-b-arrestin-1 and b-arrestin-1 expression levels in 3T3-L1 adipocytes q Yasuhito Ohsaka a,c,⇑, Hoyoku Nishino b,c a

Department of Pharmacology, Faculty of Pharmaceutical Sciences, Chiba Institute of Science, 15-8 Shiomi-cho, Choshi, Chiba 288-0025, Japan Ritsumeikan Global Innovation Research Organization, Ritsumeikan University, 1-1-1 Nojihigashi, Kusatsu, Shiga 525-8577, Japan c Department of Biochemistry and Molecular Biology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, 465 Kajii-cho, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto 602-8566, Japan b

a r t i c l e

i n f o

Article history: Received 7 March 2011 Accepted 12 March 2012 Available online 21 March 2012 Keywords: b-Arrestin-1 Aluminum fluoride Cooling Epinephrine Mithramycin A N-Ethylmaleimide Okadaic acid Phospho-Ser-412 b-arrestin-1 3T3-L1 adipocytes Receptor signaling

a b s t r a c t Cooling induces several responses that are modulated by molecular inhibitors and activators and receptor signaling. Information regarding potential targets involved in cold response mechanisms is still insufficient. We examined levels of the receptor-signaling mediator b-arrestin-1 and phospho-Ser-412 b-arrestin-1 in 3T3-L1 adipocytes exposed to 4–37 °C or treated with some molecular agents at 37 °C. We also cooled cells with or without modification and signal-modulating agents. These conditions did not decrease cell viability, and western blot analysis revealed that exposure to 4 °C for 1.5 h and to 28 and 32 °C for 24 and 48 h increased phospho-b-arrestin-1 and b-arrestin-1 levels and that exposure to 4 and 18 °C for 3 and 4.5 h increased b-arrestin-1 level. Serum removal and rewarming abolished b-arrestin-1 alterations induced by cooling. Mithramycin A (a transcription inhibitor) treatment for 4 and 24 h increased the level of b-arrestin-1 but not that of phospho-b-arrestin-1. The level of phospho-b-arrestin-1 was increased by okadaic acid (a phosphatase inhibitor), decreased by epinephrine and aluminum fluoride (receptor-signaling modulators), and unaffected by N-ethylmaleimide (an alkylating agent) at 37 °C. N-Ethylmaleimide and the receptor-signaling modulators did not alter b-arrestin-1 expression at 37 °C but impaired the induction of phospho-b-arrestin-1 at 28 and 32 °C without affecting the induction of b-arrestin-1. We show that cold-induced b-arrestin-1 alterations are partially mimicked by molecular agents and that the responsive machinery for b-arrestin-1 requires serum factors and N-ethylmaleimide-sensitive sites and is linked to rewarming- and receptor signaling-responsive machinery. Our findings provide helpful information for clarifying the cold-responsive machinery for b-arrestin-1 and elucidating low-temperature responses. Ó 2012 Elsevier Inc. All rights reserved.

Introduction Several responses to cooling are induced by the binding of ligands to their cell-surface receptors; these ligands include insulin (a glycolipid hormone) [28,36,25] and lipopolysaccharides Abbreviations: AMPK, AMP-activated protein kinase; cAMP, cyclic adenosine 30 ,50 -monophosphate; ERK, extracellular signal-regulated kinase; 4E-BP1, eIF4Ebinding protein 1; Gs, stimulatory GTP-binding protein; GTP, guanosine triphosphate; HEK, human embryonic kidney; IGF-I, insulin-like growth factor I; IRAP, insulin-responsive aminopeptidase; LPA, lysophosphatidic acid; MAPK, mitogenactivated protein kinase; MDM2, murine double minute 2; MEK, MAPK/ERK kinase; MEKK1, MAPK/ERK kinase kinase 1; PP2A, protein phosphatase type 2A; TNF-a, tumor necrosis factor-a; TRAF2, TNF receptor-associated factor 2. q Statement of funding: This work was partially supported by Grants-in-Aid from the Ministry of Education, Science and Culture of Japan, and from ProBRAIN. ⇑ Corresponding author at: Department of Pharmacology, Faculty of Pharmaceutical Sciences, Chiba Institute of Science, 15-8 Shiomi-cho, Choshi, Chiba 288-0025, Japan. Fax: +81 0479 30 4673. E-mail addresses: [email protected], [email protected] (Y. Ohsaka).

0011-2240/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.cryobiol.2012.03.001

(bacterial membrane components) [29]. The energy source glucose is transported by receptor signaling involving insulin [28] and endothelin-1 (ET-1; a vasoactive mediator) [38], and the second messenger analogue dibutyryl cAMP is an inducer of GTP-binding protein-coupled receptor (GPCR) responses. The addition of these molecules that are responsive to receptor ligands and addition of ligand agents [9], including insulin and ET-1 antagonists, to a cold preservation solution prevents hypofunction caused by cooling. Low temperatures, such as 4–32 °C, are used for organ preservation and a multitude of other applications, including clinical treatments for inflammatory conditions and tissue injuries and laboratory experiments to determine the subcellular distribution of molecules such as receptors. However, the effect of cooling on the machinery related to responses that are modulated by receptor signaling alterations is poorly understood. b-Arrestin-1 is a scaffold/adaptor protein and plays a role in desensitization, i.e., alterations in receptor sequestration and receptor signaling (e.g., receptor signaling for cAMP accumulation and

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insulin-receptor substrate (IRS) expression), caused by an agonist for the GPCR b-adrenergic receptor (b-AR) [19] and insulin [35]. bArrestin-1 also mediates the transduction of receptor signals by agonists, including ET-1 [14], IGF-I [8], and TNF-a [16]. In rat adipocytes treated with a b-AR agonist, the number of receptor ligandbinding sites for [3H]-CGP12177 (a labeled ligand that binds to bARs) on the cells cooled at 6 °C for 3 h is not markedly reduced compared with that on cells exposed to 37 °C [18]. Exposure of insulinor ET-1-treated 3T3-L1 (L1) adipocytes (differentiated preadipocytes) to cooling and subsequent binding of a labeled ligand to the cell-surface receptors at 4 °C for 64 h has been used to examine the subcellular distribution of receptor agonist-responsive molecules or receptor molecules [38]. Treatment with the b-AR agonist isoproterenol (ISO; a lipolytic agonist) decreases phosphorylation of b-arrestin-1 at the Ser-412 residue in L1 adipocytes [12]. The phosphorylation of Ser-412 b-arrestin-1 is increased by treatment of L1 adipocytes with insulin [12,13], and the production of phospho-Ser-412 b-arrestin-1 stimulated by insulin is decreased by treatment with a MEK inhibitor in L1 adipocytes [13]. Insulin treatment can also decrease the level of b-arrestin-1 expression in L1 adipocytes [12]. The influence of low temperatures on the agonistresponsive machinery for b-arrestin-1 remains unclear. Exposure of L1 adipocytes, incubated in the presence or absence of serum, to cooling at 4–32 °C induces molecular alterations [28,37,36,23–25], including changes in receptor signaling molecules (e.g., AMPK [24]), without affecting viability of the cells; subsequent rewarming decreases these responses [24,25]. Exposure to cooling at 19 °C [28] and 20 °C [37] decreases the number of molecules secreted from L1 adipocytes into the extracellular medium; the genes of some of these molecules have an a transcription factor Sp1-binding element in their promoter regions. The cellular alterations of AMPK produced by cooling at 4–32 °C for 1.5–4.5 and 24 h are similarly induced by treatment of L1 adipocytes with molecular inhibitors and activators, including mithramycin A (a transcription factor Sp1-binding inhibitor) and aluminum fluoride (a receptorsignaling modulator), at 37 °C for 4 h, 24 h, or 15 min [24]. Phospho-Ser-412 b-arrestin-1 is induced in L1 adipocytes by treatment with okadaic acid (an inhibitor of phosphatases including PP2A) for 4 h [13]. In L1 adipocytes, this inhibitor can partially mimic the cold-inducible responses of glucose transporter (GLUT) distribution and glucose transport, which are stimulated by agonists, such as insulin [28,36] and ET-1 [38]. Treatment of rat adipocytes, or adipocyte homogenates, with N-ethylmaleimide (NEM; a sulfhydrylalkylating agent) at a temperature of 23 or 30 °C decreases the insulin response of glucose transport [6] and the molecular activity of glycerol phosphate acyltransferase (GPAT) [27]. NEM treatment also modulates molecular activity, including that of PP2A [40] and MEKK1 [5]. Treatment with adrenaline, a lipolytic hormone that binds to a-adrenergic and b-adrenergic GPCRs, decreases GPAT activity in rat adipocytes [31]. Exposure of patients to mild perioperative hypothermia decreases their mean skin temperature to 32–33 °C and increases their plasma adrenaline levels [10]. Research has been conducted on the inhibitory effect of an inhibitor specific for molecules on cellular responses induced in coolingexposed L1 adipocytes [25]. However, there is still insufficient information regarding potential targets of the machinery that can be used to elucidate the mechanism of cold responses for molecules responsive to receptor agonists in adipocytes. Adipose tissues need to be repeatedly implanted because of the unpredictable and irregular absorption of the graft in plastic and reconstructive surgery. To avoid repeated liposuction, implantation has been attempted using differentiated preadipocytes; freeze-preserved differentiated preadipocytes are successfully developed for adipose tissue formation [17]. In the present study, in order to investigate low-temperature responses and molecular machinery for b-arrestin-1 in L1 adipocytes, a model cell line of

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adipose cells, we examined the levels of phospho-Ser-412 b-arrestin-1 and b-arrestin-1 in the cells exposed to different temperatures of 4–37 °C in culture medium with serum or incubated in culture medium without serum at a low temperature. We also examined b-arrestin-1 alterations in L1 adipocytes treated with mithramycin A, aluminum fluoride, epinephrine (adrenaline), or okadaic acid at 37 °C and the effects of N-ethylmaleimide and receptor-signaling modulators on b-arrestin-1 alterations induced at a mildly cold temperature. Materials and methods Materials Dulbecco’s modified Eagle’s medium (DMEM) and phosphatebuffered saline (PBS) were purchased from Nissui Pharmaceutical Co. (Tokyo, Japan). Dexamethasone, epinephrine, 3-isobutyl-1methylxanthine (IBMX), mithramycin A, N-ethylmaleimide, and sodium dodecyl sulfate (SDS) were purchased from Sigma–Aldrich Co. (St. Louis, MO, USA). Insulin was obtained from Roche Diagnostics (Indianapolis, IN, USA). Trypan blue was purchased from Invitrogen (Carlsbad, CA, USA). Aluminum hydroxide, calcium chloride, 3-(4, 5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT), fatty acid-free bovine serum albumin (BSA), magnesium sulfate, oil red O, okadaic acid, polyacrylamide, potassium chloride, sodium chloride, and sodium fluoride were purchased from Nacalai Tesque (Kyoto, Japan). Culture of 3T3-L1 adipocytes L1 cells (3T3-L1 preadipocytes; the Japanese Collection of Research Bioresources, Osaka, Japan) were cultured in DMEM supplemented with 10% heat-inactivated fetal calf serum (FCS; HyClone, South Logan, UT, USA) in a humidified atmosphere of 5% CO2 and 95% air at 37 °C. The cells were plated at a density of 3.5  104 cells/cm2 and differentiated into adipocytes, as previously described [30], in culture medium containing 0.5 mmol/L IBMX, 0.25 lmol/L dexamethasone, and 0.5 lmol/L insulin. Differentiation was confirmed by staining triacylglycerol droplets in the cells with oil red O [30]. L1 adipocytes at days 7–9 after the onset of differentiation were used for all experiments. For exposure to different temperatures, L1 adipocytes were transferred from a 37 °C incubator to a 4, 18, 28, 32, or 37 °C incubator and cultured for 1.5–4.5, 24, or 48 h in culture medium containing serum or for 1.5–4.5 h in culture medium without serum in order to exclude the possible influence of serum. Some cells that were incubated at 4 °C for 3 h were transferred back to a 37 °C incubator for 1 h to warm them after the cold exposure. For treatment with molecular inhibitors and modulators, L1 adipocytes were cultured for 4 and 24 h at 37 °C with mithramycin A or okadaic acid and for 24 h at 28 and 37 °C with or without N-ethylmaleimide in serum-containing culture medium and incubated for 15 min at 37 and 32 °C with or without aluminum fluoride or epinephrine in Krebs-Ringer HEPES buffer (2.5 mmol/L CaCl2, 10 mmol/L HEPES pH 7.4, 4.7 mmol/L KCl, 1.25 mmol/L MgSO4, and 118 mmol/L NaCl) containing 1% BSA. Measurement of protein content Protein content was measured by using a bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific, Inc., Rockford, IL). Western blot analysis Proteins were extracted from L1 adipocytes exposed to the indicated treatments and were denatured by boiling, as described

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previously [24]. Proteins (10 lg) were separated by 10% SDS–polyacrylamide gel electrophoresis and transferred onto a poly(vinylidene difluoride) (PVDF) membrane (Millipore, Bedford, MA, USA). The proteins blotted on the membrane were incubated for 1.5 h with an anti-phospho-b-arrestin-1 (Ser-412) mouse antibody (1:1000; Cell Signaling Technology, Inc., Danvers, MA, USA), antib-arrestin-1 (K-16) goat antibody (1:750; Santa Cruz Biotechnology, Santa Cruz, CA, USA), or anti-MDM2 (C-18) rabbit antibody (1:500; Santa Cruz Biotechnology). After incubating the membrane with an anti-mouse or anti-rabbit (Cell Signaling Technology) or anti-goat (Santa Cruz Biotechnology) immunoglobulin (Ig)G horseradish peroxidase (HRP)-linked antibody, the immune complexes were detected on X-ray film (Kodak X-Omat AR; Eastman-Kodak Co., Rochester, NY, USA) by using enhanced chemiluminescence (ECL) western blotting detection reagents (GE Healthcare Life Sciences, Piscataway, NJ, USA). The blots detected on the film were quantified by using the National Institutes of Health (NIH) Image version 1.61 program (http://rsb.info.nih.gov/nih-image/), and the relative intensities of the blots were normalized as values for each cell. Measurement of cell viability Nucleotides were isolated from L1 adipocytes exposed to the indicated temperatures by using a nucleotide-releasing buffer provided in the ApoSENSOR ADP/ATP Ratio Assay Kit (BioVision Research, Mountain View, CA, USA). A high ratio of ADP/ATP nucleotides is observed with cell death [2]. The ADP/ATP ratio in L1 adipocytes was determined in a luminometer (Monolight 2010; Analytical Luminescence Laboratory, San Diego, CA, USA) according to the manufacturer’s protocol. MTT formazan (1-(4,5dimethylthiazol-2-yl)-3,5-diphenylformazan) is formed by the cleavage of MTT in a reaction catalyzed by succinic dehydrogenase in the mitochondrial respiratory chain. L1 adipocytes exposed to different temperatures were also incubated with 5 mg/mL MTT for 2 h at 37 °C in culture medium. These cells were lysed after washing with PBS, and the amount of MTT formazan produced by the cleavage reaction was determined by a spectrophotometric assay as described previously [24]. The viability of cells treated with the indicated agents at 37 °C or cells cooled with or without a molecular agent at a mildly cold temperature was estimated by trypan blue exclusion tests, which detect nonviable cells as they do not exclude the dye incorporated into cells and are stained. The treated cell dishes and sham-treated cell-free dishes were incubated in a 0.2% trypan blue solution for 10 min at 37 °C and washed. Subsequently, the amount of dye present was determined spectrophotometrically at 590 nm as described previously [34]. The absorbance values obtained from the sham-treated dishes were subtracted from those obtained from the treated cells, and these values were used to assess cell viability. Statistical analysis Comparisons among multiple groups were performed by using analysis of variance (ANOVA) with Scheffe’s post-hoc test, and differences between two groups were evaluated by using unpaired Student’s t test. Statistical significance was defined as P < 0.05. Results Alterations in the levels of phospho-Ser-412 b-arrestin-1 and barrestin-1 in L1 adipocytes exposed to cooling We examined the levels of phospho-Ser-412 b-arrestin-1 and barrestin-1 in L1 adipocytes exposed to different temperatures. In

the culture medium without serum, the level of phospho-b-arrestin-1 did not change significantly in L1 adipocytes cultured at 4 and 37 °C for 1.5–4.5 h (Fig. 1A). b-Arrestin-1 expression was reduced following exposure to 37 and 4 °C for 1.5–4.5 h (Fig. 1B). In serum-containing medium, the levels of phospho-b-arrestin-1 and b-arrestin-1 increased in cells exposed to 4 °C for 1.5 h (Fig. 1C and D). In cells exposed to 4 °C for 3 and 4.5 h, the increased level of phospho-b-arrestin-1 decreased (Fig. 1C), but the increased level of b-arrestin-1 expression did not decrease (Fig. 1D). Exposure of L1 adipocytes to rewarming at 37 °C for 1 h after cooling at 4 °C for 3 h decreases the level of phospho-Thr172 AMPK that was increased by cooling [24]. The increased level of b-arrestin-1 in cells exposed to 4 °C for 3 h was decreased by exposure to rewarming at 37 °C for 1 h after cooling (Fig. 1G). In cells exposed to 18 °C for 3 h, the level of phospho-b-arrestin-1 did not increase (Fig. 1E), but the level of b-arrestin-1 increased (Fig. 1F). Exposure of L1 adipocytes to 28 and 32 °C for 24 h increased the levels of both phospho-b-arrestin-1 and b-arrestin-1 (Fig. 2A and B). These increased levels were maintained with exposure of cells to 28 and 32 °C for 48 h (Fig. 2C and D). MDM2 expression was not affected by exposure of L1 adipocytes to 28 and 32 °C for 24 h (Fig. 2E) and 48 h (Fig. 2F). These temperature conditions did not change the cellular ratio of ADP/ATP dinucleotides or the amount of MTT formazan formed by MTT cleavage in the cells (Fig. 3; some data not shown); exposure of cells in serum-free medium to 4 °C for 4.5 h changed the nucleotide ratio (Fig. 3A), but the ADP/ATP ratio and MTT formazan formation were unaffected in cells cultured in serum-containing medium at 4 °C for 4.5 h (Fig. 3A; data for MTT cleavage not shown). Changes in the levels of phospho-Ser-412 b-arrestin-1 and b-arrestin1 in L1 adipocytes treated with molecular inhibitors and modulators We treated L1 adipocytes with mithramycin A, aluminum fluoride, epinephrine, or okadaic acid and examined the phospho-Ser412 b-arrestin-1 and b-arrestin-1 levels; there was no decrease in cell viability (no change in dye staining with trypan blue in cells) following these treatments. Treatment of L1 adipocytes with mithramycin A for 4 h at 37 °C increased the level of b-arrestin-1 (Fig. 4A) but did not increase the level of phospho-b-arrestin-1 (Fig. 4B). Similar results were obtained by treatment with mithramycin A for 24 h (Fig. 4C and D). Treatment of L1 adipocytes incubated at 37 °C with epinephrine or aluminum fluoride for 15 min decreased the level of phospho-Ser-412 b-arrestin-1 (Fig. 5A and B) but did not decrease the level of b-arrestin-1 (Fig. 5C). The level of phospho-b-arrestin1 is increased in L1 adipocytes treated with okadaic acid for 4 h and in SV40 small t antigen (leading to the inhibition of PP2A activity)-expressing HIRc-B (rat-1 fibroblasts overexpressing the insulin receptor (IR)) cells [13]. An increased level of phospho-b-arrestin-1 was induced by treatment of L1 adipocytes with okadaic acid for 4 and 24 h at 37 °C (Fig. 4E; data for 4 h not shown). Effects of molecular modification and modulation agents on coldinduced b-arrestin-1 alterations In order to determine whether L1 adipocytes have the regulatory machinery for cold responses and the responsive machinery for receptor signaling at low temperatures, we examined the effect of N-ethylmaleimide (NEM) on the induction of phospho-b-arrestin1 and b-arrestin-1 in cells exposed to 28 °C for 24 h and examined the effects of epinephrine and aluminum fluoride on increased levels of phospho-b-arrestin-1 and b-arrestin-1 in cells exposed to 32 °C for 24 h. Treatment with NEM suppressed the production of phospho-b-arrestin-1 (Fig. 6A) but did not suppress the expression of b-arrestin-1 (Fig. 6B) induced by cooling; NEM treatment alone did not significantly alter the levels of phospho-b-arrestin-1 and

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Fig. 1. Effects of cooling at 4 and 18 °C on phospho-Ser-412 b-arrestin-1 and b-arrestin-1 levels in L1 adipocytes. (A–D) L1 adipocytes were cultured at 4 and 37 °C for the indicated periods in culture medium without (A and B) or with (C and D) serum. (E and F) L1 adipocytes were cultured at 18 °C for the indicated periods (E) or at 4, 18, and 37 °C for 3 h (F) in culture medium with serum. (G) L1 adipocytes were cultured at 4 and 37 °C for 3 h, and some cells were further cultured at 37 °C for 1 h in serumcontaining medium. The levels of phospho-b-arrestin-1 (A, C, and E; -p-b-arr1) and b-arrestin-1 (B, D, F, and G; -b-arr1) were determined by western blot analysis with phospho-b-arrestin-1 (Ser-412) and b-arrestin-1 antibodies, respectively. Values are presented as means ± SD of three or four experiments.  P < 0.05 versus unexposed cells cultured in serum-free medium (0 h). ⁄P < 0.05 versus cells not exposed to cooling at 4 °C (0 h). –P < 0.05 versus cells exposed to 37 °C for 3 h.

b-arrestin-1. These treatment conditions did not alter the cellular staining of trypan blue dye (Fig. 6C). Treatment with epinephrine and aluminum fluoride for 15 min decreased the increased level of

phospho-b-arrestin-1 (Fig. 5A and B) but did not decrease the increased level of b-arrestin-1 induced by cooling at 32 °C for 24 h (Fig. 5D).

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Fig. 2. Effects of cooling at 28 and 32 °C on phospho-Ser-412 b-arrestin-1 and b-arrestin-1 levels in L1 adipocytes. L1 adipocytes were cultured at 28, 32, and 37 °C for 24 h (A, B, and E) or 48 h (C, D, and F) in serum-containing medium. The levels of phospho-b-arrestin-1 (A and C; -p-b-arr1), b-arrestin-1 (B and D; -b-arr1), and MDM2 (E and F; MDM2) were determined by western blot analysis with phospho-b-arrestin-1 (Ser-412), b-arrestin-1, and MDM2 antibodies, respectively. Values are presented as means ± SD of four experiments. ⁄P < 0.05 versus cells cultured at 37 °C for 24 h. –P < 0.05 versus cells cultured at 37 °C for 48 h.

Discussion Cellular alterations in glucose transport, molecular distribution, and production of phospho-Thr-172 AMPK, phospho-Ser-166 MDM2, and phospho-Thr-308 Akt are induced upon exposure of L1 adipocytes to cooling at 4, 18–19, 28, or 32 °C for 64 or 24 h [28,36,23–25]. These effects are observed in L1 adipocytes treated with receptor agonists, including insulin [28,36,25]. The level of phospho-Ser-412 b-arrestin-1 is increased in L1 adipocytes after treatment with insulin for 1, 6, and 12 h and is decreased after treatment with ISO or LPA for 15 min [12,13]. b-Arrestin-1 expression was lower after incubation in the absence of serum at 37 °C (Fig. 1B). Treatment of L1 adipocytes with receptor agonists of ET-1, IGF-I, insulin, ISO, and TNF-a for 2–15 min alters the level of b-arrestin-1 complexed with the receptor of endothelin type A [14], IGF-I, insulin [8], or b-AR [7]; Src [7]; PP2A [13]; or TRAF2

[16]. In HEK 293 cells expressing the S412D b-arrestin-1 mutant (which mimics phosphorylated b-arrestin-1), ISO treatment does not alter binding of the b-AR ligand [I125]-cyanopindolol to the cell-surface receptor [19], and this binding is decreased in ISOtreated HEK 293 cells expressing wild-type b-arrestin-1 or the S412A b-arrestin-1 mutant (which mimics dephosphorylation) [19]. In this study, we found that exposure of L1 adipocytes to cooling altered the phospho-molecule and molecule expression levels of the scaffold/adaptor protein b-arrestin-1, which undergoes molecule modification, expression alteration, and complex formation and can modulate ligand binding induced by cell-surface receptor signaling. Incubation of L1 adipocytes in a serum-containing medium at 4 °C for 4.5 h alters the levels of MDM2 expression and phosphoSer-166 MDM2 [25], and incubation of L1 adipocytes in a serumcontaining medium at 28 and 32 °C for 24 h increases the levels

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Fig. 3. Viability of L1 adipocytes exposed to various low temperatures. (A) L1 adipocytes were exposed to 4 and 37 °C for the indicated periods in culture medium with (dotted column) or without (open column) serum, and the cellular ratio of ADP/ATP nucleotides was determined. Data are expressed as values relative to those obtained from untreated cells (0 h). (B) L1 adipocytes were exposed to 28, 32, and 37 °C for 0 h (open column), 24 h (dotted column), and 48 h (hatched column). The amount of MTT formazan formed by MTT cleavage was determined. Data are expressed as percentages of values obtained from untreated cells (0 h). Values are presented as means ± SD of three experiments. ⁄P < 0.05 versus untreated cells (0 h).

of phospho-Thr-172 AMPK and its subcellular distribution [24]. Exposure of L1 adipocytes to 28 and 32 °C for 24 h in this study also increased the level of phospho-Ser-166 MDM2 in the serum-containing medium (Fig. S1). In L1 adipocytes incubated in a serumfree buffer at 4 °C for 4 h and at 18 and 28 °C for 15 min, the levels of phospho-Thr-172 AMPK and phospho-Ser-166 MDM2 are increased [24,25]. There was no induction of phospho-b-arrestin-1 or b-arrestin-1 production in L1 adipocytes incubated in a serum-free medium at 4 or 18 °C for 1.5–4.5 h (Fig. 1A and B; data for 18 °C not shown). Exposure of HEK 293 cells to 28 °C for 1 h changes the subcellular distribution of a2C-AR [15]. Epinephrine binds to a- and b-ARs, and aluminum fluoride can modulate the signaling systems for receptors such as the adenylyl cyclase system. Treatment with NEM modifies amino acids such as cystine residues in proteins and alters protein phosphatase and kinase activities [40,5]. When L1 adipocytes were exposed to rewarming at 37 °C after cooling at 4 °C, the increased level of b-arrestin-1 decreased rapidly to the basal level (Fig. 1G), similar to the decrease in the level of phospho-Thr-172 AMPK observed by rewarming after cooling [24]. In contrast, a decrease in the level of phospho-Thr-308 Akt to the basal level is not observed under the same condition [23]. None of the treatment conditions used in this study reduced cell survival rates (some data not shown). In the

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Fig. 4. Effects of molecular inhibitors on b-arrestin-1 and phospho-b-arrestin-1 levels in L1 adipocytes. L1 adipocytes were cultured with the indicated concentrations of mithramycin A (A–D) or indicated concentrations of okadaic acid (E) at 37 °C for 4 h (A and B) and 24 h (C–E). The levels of b-arrestin-1 (A and C; -b-arr1) and phospho-b-arrestin-1 (B, D, and E; -p-b-arr1) were determined by western blot analysis with b-arrestin-1 and phospho-b-arrestin-1 (Ser-412) antibodies, respectively. Values are presented as means ± SD of three or four experiments.  P < 0.05 versus cells cultured without mithramycin A at 37 °C for 4 h. ⁄P < 0.05 versus cells cultured without mithramycin A or without okadaic acid at 37 °C for 24 h.

present study, the regulatory machinery of cold-induced alterations for b-arrestin-1 requires factors responsive to serum and is modulated by receptor signaling, including that for a- and b-ARs, and influenced through molecular sites sensitive to NEM and by

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Y. Ohsaka, H. Nishino / Cryobiology 65 (2012) 12–20

A

A

–p-β-arr1

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(μmol/L)

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(μmol/L)

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200

Fig. 5. Effects of epinephrine and aluminum fluoride on phospho-b-arrestin-1 and b-arrestin-1 levels in L1 adipocytes not exposed or exposed to cooling. L1 adipocytes were cultured at 37 °C (A–C) and 32 °C (A, B, and D) for 24 h in culture medium and further incubated with the indicated concentration of epinephrine (A, C, and D) or aluminum fluoride (B–D) for 15 min in KRH buffer containing 1% BSA. The levels of phospho-b-arrestin-1 (A; -p-b-arr1 and B) and b-arrestin-1 (C and D; b-arr1) were determined by western blot analysis with phospho-b-arrestin-1 (Ser412) and b-arrestin-1 antibodies, respectively. Blots show representative results of three experiments (A, C, and D). Values are presented as means ± SD of four experiments (B). §,⁄P < 0.05 versus cells cultured without aluminum fluoride at 37 °C.  P < 0.05 versus cells cultured without aluminum fluoride at 32 and 37 °C.

rewarming; the exposure of L1 adipocytes to different temperatures of 4–37 °C simultaneously alters the expression of MDM2, the production of phospho-MDM2, -AMPK, and -Akt, and the subcellular distribution of phospho-AMPK without altering cell viability. Inconsistencies in the responses of phospho-b-arrestin-1 and b-arrestin-1 induction were observed when L1 adipocytes were exposed to 4 °C (Fig. 1C and D) and 18 °C (Fig. 1E and F) and when L1 adipocytes were treated with mithramycin A (Fig. 4A–D), aluminum fluoride, and epinephrine (Fig. 5A–C) at 37 °C. b-Arrestin-1 can form a molecule complex with a GTP-binding protein subunit [39], GPCR kinase 5 (GRK5) [1], MEK1 [21], or PP2A [13], and GRK5 and ERK induce the phosphorylation of b-arrestin-1 in vitro [1]. Disruption of the b-arrestin-1 complex with MEK1 by using MEK displacement peptides induces dephosphorylation of b-arrestin-1 at Ser-412 in HEK 293 cells [21]. The varied b-arrestin-1 responses induced by exposure to cooling and treatment with molecular

Trypan blue staining (% of control)

1.25 (mmol/L) 150

100

50

0

(μmol/L)

Fig. 6. Effects of N-ethylmaleimide on phospho-b-arrestin-1 and b-arrestin-1 induction in L1 adipocytes exposed to cooling. (A and B) L1 adipocytes were cultured at 28 and 37 °C for 24 h (A and B) with or without the indicated concentrations of N-ethylmaleimide (NEM). The levels of phospho-b-arrestin-1 (A; p-b-arr1) and b-arrestin-1 (B; -b-arr1) were determined by western blot analysis with phospho-b-arrestin-1 (Ser-412) and b-arrestin-1 antibodies, respectively. (C) L1 adipocytes treated as described in 6A and 6B were stained with trypan blue, and the amount of dye staining was determined. Data are expressed as percentages of values obtained from untreated cells (0 h). Values are presented as means ± SD of three or four experiments. ⁄P < 0.05 versus cells cultured without N-ethylmaleimide at 37 °C for 24 h.  P < 0.05 versus cells cultured without N-ethylmaleimide at 28 °C for 24 h.

agents may be due to alterations in the activities of protein kinase and phosphatase molecules such as GRK5, ERK, and PP2A. Treatment of L1 adipocytes with mithramycin A for 24 h increases the level of phospho-Thr-172 AMPK [24] and decreases

Y. Ohsaka, H. Nishino / Cryobiology 65 (2012) 12–20

the effect of a receptor agonist on the gene expression of resistin (an adipokine) [4]. In addition, treatment of L1 adipocytes with mithramycin A for 1 h decreases the effect of ET-1 on transcriptional activation of interleukin-6 (a cytokine) [3]. Furthermore, treatment of L1 adipocytes with aluminum fluoride for 15 min alters the subcellular distribution of phospho-Thr-172 AMPK [24], treatment with epinephrine for 0.25–1 h induces molecular release of glycerol, and treatment with okadaic acid for 0.5 h increases phosphorylated levels of IR and IRS [32]. Treatment with both okadaic acid and insulin for 6 h does not increase the level of phospho-Ser-412 b-arrestin-1 above the level induced by insulin alone in L1 adipocytes [13]. Cold responses for alterations in b-arrestin-1 and phospho-b-arrestin-1 levels were similarly induced by mithramycin A and okadaic acid and partially mimicked by receptor-signaling modulators. These molecular agents are able to alter phospho-molecule production, receptor-mediated responses, and phospho-molecule distribution in L1 adipocytes, which include pathways for changes in the expression and transcription of genes, including adipokine and cytokine genes, and for inducing the distributed and released molecules and the phosphorylated molecules of the receptor, its substrate, and the protein kinase. Treatment with anisomycin, which induces protein kinase activation and translation inhibition, and treatment with cobalt chloride, which modulates molecular expression, produce several responses similar to those stimulated by cooling. In L1 adipocytes, treatment with anisomycin for 4 and 24 h and treatment with cobalt chloride for 24 h induce the production of phospho-Thr-172 AMPK, which is stimulated by cooling at 4–32 °C, [24]. Treatment of L1 adipocytes with anisomycin produces cellular responses, including glucose transport, p38 MAPK activation, and phospho-molecule production of a range of targets, including c-Jun, IRS, and p70 S6 kinase. Cobalt chloride treatment modulates the expression levels of molecules, such as adipokines and transcription factors, in L1 adipocytes. The b-arrestin-1 alterations induced by cooling at 4–32 °C were also mimicked by treatment of L1 adipocytes with anisomycin for 4 and 24 h at 37 °C (Fig. S2-A and B) and treatment with cobalt chloride for 24 h at 37 °C (Fig. S2-C). Molecular agents that induce cold responses are used for examination of the mechanism by which molecular alterations are induced at low temperatures. The genes of some of the coldresponsive molecules in L1 adipocytes have a transcription factor Sp1-binding element in their promoter regions. The ability of mithramycin A to inhibit the protein expression of MDM2 is affected by a sequence polymorphism found close to the Sp1-binding site in the MDM2 promoter. In L1 adipocytes, treatment with mithramycin A decreases the expression level of MDM2 [24,25]. Further inhibitory experiments targeting molecules sensitive to Sp1 in the b-arrestin-1-related machinery responsive to cold are required to elucidate the role of these molecules in low-temperature responses. Exposure to 20–25 °C for 90 min slightly decreases the cellular phosphatase activity in NF9006 mammary carcinoma cells [22]. PP2A is present in molecular complexes immunoprecipitated with the b-arrestin-1 antibody from L1 adipocytes [13]. Additionally, exposure of L1 adipocytes to 4 °C for 3 h does not change the level of phospho-4E-BP1, which inhibits the binding of 4EBP1 to eukaryotic initiation factor 4E (eIF4E), [23]. It would be interesting to determine whether exposure of L1 adipocytes to cooling for 1.5–4.5 h and P24 h alters other molecular factors, including protein kinases and phosphatases such as PP2A, and whether these molecular alterations are involved in b-arrestin-1associated cold responses. The effects of agents mimicking low-temperature responses provide possible clues for elucidating the regulatory mechanisms of cold-induced alterations of b-arrestin-1 in L1 adipocytes. Treatment of adipocytes with insulin reverses the inhibitory effect of a- and b-AR agonists on the activity of GPAT [31,27],

19

and this GPAT activity is sensitive to NEM [27]. NEM treatment inhibits the insulin response of glucose transport in adipocytes [6] and other agonist responses in other cells, including PP2A activation and production of phospho-molecules, such as phosphoThr-308 Akt, phospho-p70 S6 kinase, and phospho-4E-BP1 [40]. Thus, treatment with NEM modulates the molecules responsive to receptor-signaling activation. The expression level of b-arrestin-1 in L1 adipocytes is decreased by treatment with insulin for 12 h [12]. In addition, expression of the b-arrestin-1 gene is increased by treatment of human umbilical vein endothelial cells (HEC) with forskolin, an adenylate cyclase activator and cAMP inducer, or iloprost, a prostacyclin analog acting through a Gs-coupled receptor, for 6 h [26]. The increased expression of barrestin-1 induced at a mildly cold temperature was not decreased by treatment of L1 adipocytes with epinephrine, aluminum fluoride (Fig. 5D), and NEM (Fig. 6B), while the increased level of phospho-b-arrestin-1 induced by cooling was decreased by these agents (Figs. 5A and B and 6A). The b-arrestin-1 regulatory machinery at a mildly cold temperature is linked to, and may involve, the responsive machinery for cell-surface receptor signaling. b-Arrestin-1 is altered by receptor–ligand signaling activation. Exposure of L1 adipocytes to different temperatures in the range of 4– 37 °C induced a b-arrestin-1 alteration that is produced by cell-surface receptor signaling. Our findings contribute to an increased understanding of low-temperature responses and their responsive machinery in L1 adipocytes. A b-arrestin-1 complex with clathrin, which is included in the formation of coated vesicles, is formed in HEK 293 cells expressing the S412A b-arrestin-1 mutant [19] and in HEK 293 cells expressing a dominant-negative MEK1 (K97A), which decreases an active form of ERK, [20]. Additionally, a b-arrestin-1 complex with b-AR is formed in ISO-stimulated HEK 293 cells that have been transfected with the S412A or S412D b-arrestin-1 mutant [19]. In L1 adipocytes, b-arrestin-1 binds to several receptors [8,14] and protein kinase and phosphatase molecules [7,14,13]. Treatment of HIRc-B cells with insulin for 6 h decreases the expression of IRS-1 but does not decrease the expression of IRS-2 [35]. This decrease in IRS-1 expression induced by insulin is prevented by transfection of HIRc-B cells with the S412A b-arrestin-1 mutant but not by transfection with the S412D b-arrestin-1 mutant [35]. On the other hand, treatment with insulin increases the expression of IRS-2 in HIRc-B cells transfected with S412A or S412D b-arrestin-1 mutants [35]. IRS-1 and -2 proteins respond to insulin in L1 adipocytes, resulting in, for example, the production of phospho-IRS-1 and phospho-IRS-2. In L1 adipocytes incubated at 4, 19 and 23 °C, insulin treatment for 15 min and 1 h induces the production of phospho-IRS-1 [11] and insulin treatment for 0.5 and 1.5 h alters the subcellular distribution of EGFP-PH/ARNO [36] (which binds to inositol phospholipids) and GLUT4/IRAP [33]. The inflammatory cytokine TNF-a is released upon exposure of human blood peripheral mononuclear cells to cooling at 32 °C for 48 h [29]. b-Arrestin-1 forms a molecule complex with TRAF2 in L1 adipocytes [16], and the formation of this complex is decreased in TNF-a-treated L1 adipocytes [16]. Exposure of L1 adipocytes to cooling might change phosphob-arrestin-1 and b-arrestin-1 levels in order to interact with other molecules that are involved in receptor-responsive machinery and to modulate molecular responses that are induced by alterations in receptor signaling. Further studies are needed to elucidate the physiological relevance of cold-responsive b-arrestin-1 alterations and the significance of these responses in L1 adipocytes. Acknowledgments This work was partially supported by Grants-in-Aid from the Ministry of Education, Science and Culture of Japan, and from ProBRAIN.

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Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.cryobiol.2012. 03.001.

[20]

[21]

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