Molecular Identification and Characterization of a Novel Nuclear Protein Whose Expression Is Up-regulated in Insulin-resistant Animals*

Energy metabolism is the most fundamental capacity for mammals, impairment of which causes a variety of diseases such as type 2 diabetes and insulin resistance. Here, we identified a novel gene, termed diabetes-related ankyrin repeat protein (DARP) that is up-regulated in the heart of KKAy mouse, a type 2 diabetes and insulin resistance model animal. DARP contains putative nuclear localization signals and four tandem ankyrin-like repeats. Its expression is restricted in heart, skeletal muscle, and brown adipose. Western blot analysis and immunocytochemistry of DARP-transfected Chinese hamster ovary (CHO) and COS-7 cells reveal that DARP is a nuclear protein. When DARP is expressed in CHO cells, [1-14C]palmitate uptake is significantly decreased, whereas the palmitate oxidation does not show significant change. Furthermore, DARP expression is altered by the change of energy supply induced by excess fatty acid treatment of skeletal myotube in vitro and fasting treatment of C57 mouse in vivo. We confirmed that DARP expression is also altered in Zucker fatty rat, another insulin resistance model animal. Taken together, these data suggest that DARP is a novel nuclear protein potentially involved in the energy metabolism. Detailed analysis of DARP may provide new insights in the energy metabolism.

Metabolic disorders cause wide variety of diseases including hyperlipidemia, hyperuricaemia, diabetes, and insulin resistance. Among these diseases, diabetes and insulin resistance are epidemic worldwide and are expected to affect 300 million people by 2025 (1). Recently, abnormalities of fatty-acid metabolism are recognized as key components of the pathogenesis of type 2 diabetes and insulin resistance (2)(3)(4)(5). High fat diet and raised levels of circulating free fatty acids are sufficient to induce insulin resistance that is related to the fat content of skeletal muscle in rats (6). Accumulation of lipids inside muscle cells and, specifically, an increase in muscle long chain fatty acyl-CoA content are reported to cause insulin resistance. This suggests that abnormal fatty acid metabolism and the accumulation of lipid in skeletal muscle play crucial roles in the pathogenesis of insulin resistance (7,8). Moreover, the relation between insulin resistance and muscle triglyceride content is independent of total adiposity. Although the details of the mechanisms connecting lipid accumulation and insulin resistance are still unclear, studies of insulin receptor signaling reveal that the accumulation of lipid products causes the phosphorylation of insulin receptor as well as insulin receptor substrate (IRS)-1 through protein kinase C activation. This results in the inhibition of insulin receptor signaling (4).
Recently identified molecules involved in the pathogenesis of insulin resistance act at least partially through the alteration of fatty acid metabolism. Adiponectin, whose secretion from white adipose tissue is reduced in insulin-resistant animal models, induces tissue fatty acid oxidation, leading to a reduction of tissue steatosis and reduced plasma glucose, triglycerides, and free fatty acids concentrations (9). The new class of insulin-sensitizing agents, thiazolidinediones, affects a wide variety of metabolic genes in insulin-sensitive tissues and has direct effects on mitochondrial fuel oxidation (10 -12).
Here, we report a novel nuclear protein, termed diabetesrelated ankyrin repeat protein (DARP), 1 that is up-regulated in the heart of KKA y mouse, a type 2 diabetes and insulin resistance model animal. DARP-expressing CHO cells demonstrate significantly decreased [1-14 C]palmitate uptake. Furthermore, DARP expression in skeletal muscle is altered by a change of energy supply both in vitro and in vivo. Also, DARP expression is altered in Zucker fatty rat, another insulin resistance model animal. These results suggest that DARP is a novel nuclear protein that is potentially involved in energy metabolism.

EXPERIMENTAL PROCEDURES
Cloning of DARP-Total RNA was extracted from the heart of 10week-old KKA y and C57BL/6J mice (Nihon Crea) using ISOGEN (Wako Pure Chemical Industries). Suppression-subtractive hybridization was performed using PCR-Select cDNA Subtraction Kit (BD Biosciences Clontech) with 2 g of poly(A) ϩ RNA purified from total RNA using the FastTrack 2.0 Kit (Invitrogen) as recommended by the manufacturers. Subtracted cDNAs were subcloned into pT7 vector (Novagen) and sequenced. The 5Ј end of DARP cDNA was cloned by 5Ј-rapid amplification of cDNA ends (5Ј-RACE) (Invitrogen) against C57BL/6J mouse heart. The primers for 5Ј-RACE were designed according to the sequences obtained by the search of GenBank TM (5Ј-TTCACCAGCT-GTCTGTGGCCCTTCAGACA-3Ј for synthesis of first strand cDNA, 5Ј-CAGGTACTTGTCAATCAGGGCCTCCTGGT-3Ј for first PCR, and * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM  5Ј-TTTAGTTTCTCCCGGGGCCACAGCCTCTT-3Ј for nested PCR). The full-length cDNA was obtained by RT-PCR as described (13). Human DARP cDNA was obtained by RT-PCR using human skeletal muscle total RNA (Sawady Technology). The primers were designed according to the sequences obtained by the search of GenBank (forward primer: 5Ј-GGACCATGGACTTCATCAGCATTCAGCA-3Ј; reverse: 5Ј-TCAGCACCGGGTGCGGGGATGCGCCACGT-3Ј). For nucleotide sequencing, overlapping restriction fragments of the cDNA were subcloned into pBluescript vector (Stratagene) and sequenced. Both strands of cDNA were covered at least twice.
Northern Blot Analysis-338-bp cDNA, corresponding to the 3Ј end of mouse DARP coding region, labeled with [␣-32 P]dCTP, was used for Northern analysis with QuickHyb solution (Stratagene) as recommended by the manufacturer. The washed blots were exposed to imaging plate analyzed by BAS analyzer (Fuji film).
Cell Culture and Transfection-Rat skeletal myoblasts were isolated from rat soleus muscles as described (14). Confluent myoblasts were differentiated into myotube by incubating in Dulbecco's modified Eagle's medium containing 2% horse serum (differentiation medium) for 4 days. Differentiated myotube were cultured for another 24 h with or without 250 M oleic acid-albumin (Sigma). CHO-K1 cells were cultured as previously described (15). Oligo nucleotides that encode a FLAG tag (DYKDDDDK) were inserted at the end of the DARP coding region by PCR-based mutagenesis (DARP-FLAG cDNA). The primers used were 5Ј-GGACCATGGACTTCATCAGCATTGAGGAG-3Ј and 5Ј-TCATTTG-TCGTCGTCGTCCTTGTAGTCGCACCGGGTACGGGGATGGCCCAC-3Ј. The DARP-FLAG cDNA was subcloned into the pME18Sf expression vector (14). Stable transfection of CHO cells and isolation of the transfectant clones were performed as described (14). Briefly, DARP-FLAG expression construct was co-transfected with pSV2neo vector into CHO cells by lipofection. 24 h after transfection, cells were seeded sparsely and incubated in the medium containing 0.1% G418 for 1 week. Isolated colonies were picked up using a cloning cup, and we confirmed the expression of DARP-FLAG mRNA and protein. Transient transfection of DARP-FLAG into COS-7 cells was performed using LipofectAMINE PLUS (Invitrogen) as recommended by the manufacturer.
Western Blot Analysis and Immunocytochemistry-For Western blot analysis, cells were lysed in the lysis buffer (1% zwittergent, 50 mM phosphate, and 150 mM NaCl), and the concentrations of which were measured by Bio-Rad Protein Assay (Bio-Rad) based on the method of Bradford using bovine serum albumin as the standard. 30 g of each protein were separated on 10% SDS-polyacrylamide gel and transferred to a polyvinylidene difluoride membrane. After blocking with TBS containing 5% (v/v) skim milk for 30 min, the membrane was incubated for 1 h with 10 g/ml anti-FLAG M2 antibody (Sigma) followed by washing with TBS containing 0.1% Tween 20 three times for 5 min each. The membrane was then incubated with horseradish peroxidase-conjugated anti-mouse IgG antibody for 1 h. After washing the membrane three times for 15 min each, signals were detected using ECL system (Amersham Biosciences). Nuclear and cytoplasmic fractions were extracted using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Pierce) as recommended by the manufacturer. 30 g of each fraction were subjected to Western blot analysis. For immunocytochemistry, cells were fixed and permeabilized in methanol for 5 min at Ϫ20°C. After washing in PBS, PBS containing 10% (v/v) normal goat serum (NGS/ PBS) was added. Following a 1-h incubation at 37°C, the NGS/PBS was replaced with buffer containing anti-FLAG M2 antibody (1:300). After incubation for 90 min at 37°C, the cells were washed with PBS three times for 10 min each and then incubated in NGS/PBS containing 1.7 g/ml of RITC-goat anti-mouse IgG antibody (EY Laboratories) for 1 h at 37°C. Following incubation, cells were washed six times with PBS for 10 min each. The coverslips were mounted on the slides with 90% (v/v) glycerol, 50 mM Tris-HCl (pH 9.0), and 2.5% (v/v) 1,4-diazabicyclo-[2.2.2]octane and observed with a confocal fluorescent microscopy.
Assay of Metabolism of [1-14 C]Palmitate-[1-14 C]Palmitate-BSA was prepared as described (16). Briefly, [1-14 C]palmitic acid dissolved in toluene (Amersham Biosciences) was incubated at 35°C under nitrogen gas to evaporate toluene. After evaporation, the palmitic acid was dissolved in water containing KOH by incubating at 40°C. Subsequently, palmitate solution was mixed well with BSA-containing solution in the ratio of 700 mg of BSA and 7.7 mg of palmitate. To examine the effect of DARP on [1-14 C]palmitate metabolism, approximately an equal number of CHO cells with or without expression of DARP-FLAG were incubated in serum-free Dulbecco's modified Eagle's medium/F-12 containing 40 M [1-14 C]palmitate for 6 h. For determination of the palmitate uptake, cells were washed three times with PBS and harvested. After collection by brief centrifugation, the cell pellet was dissolved in scintillation fluid assayed for radioactivity. For determination of palmitate oxidation, cells were cultured in the sealed flask containing a suspended filter paper. The 14 CO 2 in the medium was liberated by addition of 1 ml of 6 N hydrochloric acid. The 14 CO 2 collected overnight on the filter paper alkalized with 2 N sodium hydroxide was quantified by scintillation counting.
In Vivo Experiments-Animal care and procedures were in accordance with guidelines and regulations of the institutional animal care committee. To examine DARP expression, 8-week-old Zucker fatty and lean rats (Charles River, Japan) were used. For fasting, 8-week-old C57BL/6J mice were divided into three groups. One group was maintained on chow, a second was fasted for 48 h, and a third was fasted for 48 h followed by unrestricted access to chow for 48 h.
Statistical Analysis-All data are presented as mean Ϯ S.E. as indicated. Statistical analyses of the characteristics of Zucker rats and Northern blot analyses were performed with a Mann-Whitney's U test. Differences between mean values obtained for palmitate metabolism studies were determined by a Student's t test. p Ͻ 0.05 was considered significant.

RESULTS
Identification of DARP-In the heart, energy metabolism is appreciably active and dynamic. Alteration of heart energy metabolism is reported in diabetes and insulin resistance model animals (17,18). To isolate genes that are involved in energy metabolism, we have performed suppression-subtractive hybridization using the heart of the KKA y mouse, a model mouse of type 2 diabetes and insulin resistance. Since the KKA y mouse shows obesity and insulin resistance due to polygene impairment, there is no authentic normal control mouse with same genetic background. Although the KK mouse is used as a control for the KKA y mouse in several studies, the KK mouse shows mild diabetic phenotype. Thus, KK mouse is not an appropriate control mouse for our experiments, and we used the C57 mouse as a control.
We then successfully identified a novel gene, termed DARP, whose expression is up-regulated in KKA y mouse heart as compared with C57 mouse heart (Fig. 1). Nucleotide homology search of the GenBank and 5Ј-RACE using mouse heart total RNA allowed us to isolate a full-length DARP cDNA. DARP encodes 306 amino acids containing putative nuclear localization signals and four tandem ankyrin (ANK)-like repeats. The amino acid sequence of DARP showed high similarity to cardiac ankyrin-repeat protein (CARP) and ankyrin-repeat domain 2 (Ankrd2) with 45 and 36% identities, respectively ( Fig. 2A). We also isolated human DARP cDNA by RT-PCR using human skeletal muscle total RNA (Fig. 2B). Searching the GenBank of human DARP gene revealed that it is located at chromosome 2p11.1-2q11.1 where no genetic diseases are reported.
Tissue Distribution of DARP mRNA-Northern blot analysis of adult mouse tissues revealed that DARP is expressed most abundantly in skeletal muscle and also highly expressed in heart and brown adipose tissue (Fig. 3). All of these tissues are metabolically highly active. No signal was observed in other tissues, including brain, lung, liver, kidney, intestine, and white adipose.
Expression of DARP in Eukaryotic Cells-Since DARP contains putative nuclear localization signals, we investigated FIG. 1. DARP expression in C57 and KKA y mouse heart. 10 g of total RNA extracted from the hearts of C57 and KKA y mice were separated by electrophoresis on 1% agarose/formaldehyde gel and transferred onto a nylon membrane subjected to Northern blot analysis of DARP. The same blot was stripped and re-probed for ␤-actin mRNA.
whether DARP is indeed a nuclear protein. We prepared CHO cells that stably express DARP with FLAG tag attached at its C terminus (CHO/DARP-FLAG). Western blot analysis of the cell lysate of CHO/DARP-FLAG demonstrated appropriate DARP expression in the size of ϳ34.3 kDa, although nonspe-cific signals were observed in both CHO/DARP-FLAG and vector-transfected CHO cells (CHO/MOCK) (Fig. 4A). Immunocytochemistry of CHO/MOCK demonstrated relatively strong immunoreactivities dispersed diffusely, presumably due to nonspecific cross-reaction with anti-FLAG antibody (data not shown). We then performed Western blot analysis of nuclear and cytoplasmic fractions of CHO/DARP-FLAG as described. Appreciable amount of DARP-FLAG expression was observed in the nuclear fraction, although a significant amount of protein still remained in the cytoplasmic fraction, presumably due to ongoing protein synthesis (Fig. 4B). To further confirm its nuclear localization, we performed Western blot analysis of nuclear and cytoplasmic fractions of COS-7 cells in which DARP-FLAG was transiently transfected (COS/DARP-FLAG). In COS/DARP-FLAG cells, we also detected the appreciable amount of DARP-FLAG expression in the nuclear fraction (Fig.  4B). Immunocytochemistry of COS/DARP-FLAG cells demonstrated strong immunoreactivities in the nucleus, whereas no significant immunoreactivities were observed in vector-transfected COS-7 cells (COS/MOCK) (Fig. 4C). These findings indicate that DARP is a nuclear protein.
Effects of DARP on Fatty Acid Metabolism-Recently, evi- dence that abnormalities of fatty acid metabolism in skeletal muscle play crucial roles in the pathogenesis of insulin resistance is increasing. Abundant expression of DARP in skeletal muscle and its altered expression in the KKA y mouse led us to investigate its function in fatty acid metabolism. We prepared three individual stable transfectants of CHO cells that stably express DARP-FLAG (CHO/DARP-1, 2 and 3) and compared their [1-14 C]palmitate metabolism to that of parental CHO cells (CHO/control). The expression of DARP-FLAG mRNA and protein were confirmed by Northern blot and Western blot analysis (Fig. 5, A and B). After 6 h of incubation in medium containing 40 M [1-14 C]palmitate, palmitate uptake, measured as the amount of radioactivity in the cells, slightly but significantly decreased in all three stable transfectants that express DARP-FLAG compared with control (Fig. 5C). On the other hand, palmitate oxidation in DARP-FLAG transfectants did not show significant differences from that of control cells (Fig. 5D).
DARP Expression Is Regulated by Energy Supply-We then examined the effect of exogenous energy supply on DARP expression. In skeletal myotube, addition of 250 M oleate in differentiation medium containing glucose significantly increased DARP expression after 24 h of incubation, indicating that DARP expression is altered by exogenous energy supply in vitro (Fig. 6A). There was no significant morphological change after addition of oleate, and the expression of acetylcholine receptor ␣ subunit (AchR␣), a marker of differentiated myo-tubes, did not change. These findings suggest that addition of oleate in the medium does not affect the differentiation state of skeletal myotube. To confirm the regulation of DARP expression by energy supply, we further examined the effect of fasting on DARP expression using the C57 mouse in vivo. After 48 h of fasting, DARP expression in skeletal muscle was significantly reduced. Interestingly, 48 h of re-feeding after 48 h of fasting

FIG. 5. Effect of DARP expression on [1-14 C]palmitate metabolism.
A, total RNAs were extracted from each stable transfectant and subjected to Northern blot analysis. All three transfectants express DARP-FLAG mRNA, whereas no expression of DARP was detected in parental CHO cell. B, cell lysate of each stable transfectant was subjected to Western blot analysis using anti-FLAG M2 antibody. All three transfectants express DARP-FLAG protein. C, palmitate uptake measured as the amount of radioactivity in CHO/DARP and CHO/control cells incubated with [1-14 C]palmitate as described under "Experimental Procedures." Counts per minute was determined by scintillation counting and was normalized to the value of CHO/control cells (ϭ 100%) (n ϭ 10 for control group, and n ϭ 5 for each CHO/DARP group). Mean Ϯ S.E. are shown; *, p Ͻ 0.0005 versus CHO/control; and **, p Ͻ 0.005 versus CHO/control. D, total 14 CO 2 produced from [1-14 C]palmitate in CHO/DARP and CHO/control cells (n ϭ 9 for control group and n ϭ 5 for each CHO/DARP group). Palmitate oxidation did not show any significant difference between groups. increased DARP expression in skeletal muscle to an even higher level than that of control mice without fasting (Fig. 6B). However, fasting showed no significant effects on DARP expression in either the heart or brown adipose tissue (data not shown). These observations indicate that DARP expression is, at least partially, regulated by the energy supply in skeletal muscle both in vitro and in vivo.
DARP Expression Is Altered in Insulin Resistance Model Animals-We cannot exclude the possibility that enhanced DARP expression in KKA y mouse heart is due to the difference of genetic background between KKA y and C57 mice. Therefore, we examined DARP expression in another type 2 diabetes and insulin resistance model animal, Zucker fatty rats. Zucker fatty rats showed significantly higher body weight and plasma insulin level than those of Zucker lean control rats, whereas the blood glucose of both groups was not significantly different, FIG. 6. DARP expression is regulated by energy supply. A, 30 g of total RNA isolated from skeletal myotube incubated with or without 250 M oleate for 24 h were subjected to Northern blot analysis of DARP and AchR␣. The same blot was stripped and reprobed for ␤-actin mRNA. B, 10 g of total RNA extracted from skeletal muscle of mice with unrestricted feeding, fasted, or fasted and then refed were subjected to Northern blot analysis. Radioactivities of DARP mRNA signals were normalized with actin signal (n ϭ 5 for control and fasted group, and n ϭ 6 for fasted and then refed group). Values (mean Ϯ S.E.) are presented as a percent of control (ϭ 100%); *, p Ͻ 0.01 versus control; **, p Ͻ 0.01 versus fasted group; and ***, p Ͻ 0.05 versus control.
FIG. 7. DARP expression is altered in insulin resistance model animals. 20 g of total RNA from the heart (A) and brown adipose (C) and 5 g of total RNA from skeletal muscle (B) of Zucker fatty rats and Zucker lean rats were subjected to Northern blot analysis of DARP. The same blots were stripped and reprobed with ␤-actin probe. Radioactivities of DARP mRNA signals were normalized with actin signal. Values (mean Ϯ S.E.) are presented as a percent of lean control (ϭ 100%) (n ϭ 5 for each group); *, p Ͻ 0.05 versus lean rats; and **, p Ͻ 0.01 versus lean rats. indicating that they are appropriate model animal for insulin resistance (Table I). DARP expression in heart and skeletal muscle was significantly higher in Zucker fatty rats than Zucker lean control rats (Fig. 7, A and B). In contrast, DARP expression in brown adipose was significantly lower in Zucker fatty rats as compared with that of control rats (Fig. 7C). These results indicate that DARP expression is indeed altered in insulin resistance animals. DISCUSSION We have described the cloning and characterization of DARP, a novel nuclear protein, whose mRNA expression is altered in type 2 diabetes and insulin resistance model animals. From the data presented in this manuscript, we are unable to determine whether DARP has a clear function in free fatty acid metabolism. However, its restricted expression in heart, skeletal muscle, and brown adipose and relevance to fatty acid metabolism suggest that analysis of DARP may reveal new insights in the energy metabolism.
Amino acid sequencing of DARP revealed that it contains putative nuclear localization signals and four tandem ANKlike repeats, sharing high homology with CARP and Ankrd2. CARP was initially identified as a cytokine-inducible nuclear protein from human endothelial cells (19). Later, CARP was reported to be a downstream molecule in the Nkx2-5 homeobox gene pathway in cardiomyocyte (20) and to be a downstream target of TGF-␤/Smad signaling in vascular smooth muscle cell (21). However, its physiological function is still unclear. Ankrd2 was identified from mouse skeletal muscle as a gene putatively responsible for stretch-induced muscle hypertrophy (22,23). Their identical structural features are nuclear localization signals and the ANK repeat motif. Although ANK repeats were initially reported to mediate protein-protein interactions, their function is more diverse. ANK repeat proteins carry out a wide variety of biological activities, and this motif has been recognized in more than 400 proteins including cyclin-dependent kinase inhibitors, transcriptional regulators, cytoskeletal organizers, developmental regulators, and toxins (24). Thus, the ANK repeat motif does not determine the specific function of DARP, although it may play key roles in DARP function.
Immunocytochemistry of COS/DARP-FLAG cells and Western blot analysis of nuclear and cytoplasmic fractions of COS/ DARP-FLAG and CHO/DARP-FLAG demonstrated that DARP is a nuclear protein. Its nuclear localization suggests that DARP may play a role in the regulation of gene expression. It was reported that subcellular localization of CARP is altered by a change of circumstance of the cell, such as serum depletion in vitro (20). Since the function of protein is sometimes regulated by its subcellular localization (25,26), detailed analysis of DARP localization may provide important clues to clarify the physiological function of DARP.
DARP expression in CHO cells caused a slight but significant decrease of palmitate uptake, suggesting that DARP may be involved in fatty acid metabolism. However, in CHO cells, it appeared that the effect of DARP on fatty acid metabolism was not enhanced proportionately to its expression level. Because DARP possesses ANK-like repeats that may mediate proteinprotein interaction, DARP may functionally require partner molecule(s). Therefore, over-expression of DARP in CHO cells may not be sufficient. However, our results strongly suggest that DARP is potentially involved in fatty acid metabolism. Further experiments are required to elucidate the detailed mechanism of DARP effect on fatty acid metabolism.
Because skeletal muscle is the principal tissue for insulinmediated glucose disposal and a major site of peripheral insulin resistance, the correlation between skeletal muscle fuel handling and insulin resistance has been extensively investi-gated. Recent studies revealed that skeletal muscle in insulin resistance shows increased glucose oxidation and decreased fatty acid oxidation under basal conditions and decreased glucose oxidation and increased fatty acid oxidation under insulinstimulated conditions. This is referred to as a state of "metabolic inflexibility" (4). Decreased fatty acid oxidation under basal conditions could lead to lipid accumulation within skeletal muscle that is strongly associated with insulin resistance. Since DARP-expressing CHO cells demonstrated a significant decrease of palmitate uptake, DARP expression may be upregulated in insulin-resistant animals to partially compensate for abnormalities in fatty acid accumulation in skeletal muscle. However, further analyses are required to address this point. DARP expression is altered by a change of energy supply and energy metabolic condition, induced by excess fatty acid treatment in vitro and fasting in vivo. Initially, we expected that fasting would enhance the expression of DARP as well as excess fatty acid treatment in vitro since fasting was shown to increase plasma fatty acid level (27). Unexpectedly, fasting resulted in a decrease in DARP expression. This could be due to a significantly reduced glucose supply under fasted conditions. Although detailed mechanisms are unknown, our observations suggest that DARP expression is, at least partially, regulated by energy supply. Since energy supply appreciably affects energy metabolism (27,28), these findings further suggest that DARP is implicated in energy metabolism. To clarify the physiological function of DARP, gene targeting and transgenic animal studies will likely be required. Detailed analysis of DARP will provide new insights of energy metabolism and crucial information as to the molecular regulatory mechanisms of energy metabolism.