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RNA Extraction and Northern Blotting—Total RNA was extracted from adipose tissue (frozen immediately (0 minutes)) or after incubation in the absence (basal) or in the presence of insulin (6 nM) for 2 h using the modified method of Chomczynski and Sacchi (15). Leptin mRNA levels were detected as described previously (16).

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Transient Transfection of Reporter Constructs into Differentiated 3T3-L1 adipocytes and Luciferase Assay桹n day 4 of differentiation, 3T3-L1 adipocytes were transfected with 1 礸 of DNA using Lipofectamine Plus reagents (Invitrogen). Briefly, cells were washed with phosphate-buffered saline and incubated with reduced serum medium (Opti-MEM? modification of Eagle's minimal essential medium, Invitrogen) for 45 min at 37 癈. Pre-complex DNA-Plus-LipofectamineTM reagent was prepared according to the manufacturer's protocol and added into each well for transfection. After overnight transfection, cells were replenished with 10% fetal bovine serum Dulbecco's modified Eagle's medium and incubated for another 36?8 h for expression. For monitoring transfection efficiency, pRL-TK vector (containing Renilla luciferase, Promega) was co-transfected. Cells co-transfected with various UTR-pGL3 constructs and pRL-TK vectors were harvested in lysis buffer, and activities of firefly and Renilla luciferase were measured by using the Dual-Luciferase assay kit (Promega) in a luminometer (Turner Designs, Sunnyvale, CA). The reporter activity was expressed as arbitrary LUC units (firefly/Renilla).

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The Effects of Starvation and Insulin on Leptin Biosynthesis Are Specific, Comparison with LPL—In agreement with previous studies (14), overnight starvation did not change the rates of basal LPL biosynthesis in adipose tissue (Fig. 2C, each n = 3). These data show the decrease in leptin biosynthesis after starvation was specific and not a result of a general decline in the biosynthesis of adipose-specific genes or due to an artifact related to alterations in the specific activity of the [35S]Met/Cys precursor pool. Additionally, insulin increased the relative rates of LPL biosynthesis both in starved (+72 ± 13%, p < 0.05, n = 3) and in fed tissue (+66 ± 21%, p < 0.05, n = 3) but to a lesser extent than leptin.

J. Biol. Chem., Vol. 282, Issue 1, 287-293

Recombinant Adenovirus Transduction—The mutant Foxo1 carries single amino acid substitutions replacing the three main phosphorylation sites, Thr24 " src="http://www.jbc.org/math/rarr.gif" border=0> Ala, Ser253 " src="http://www.jbc.org/math/rarr.gif" border=0> Asp, and Ser316 " src="http://www.jbc.org/math/rarr.gif" border=0> Ala, and has been described previously (29). INS832/13 cells were seeded 2 days before use and transduced with adenovirus encoding Foxo1 or -galactosidase at a multiplicity of infection of 50 plaque-forming units/cell for 1 h in 1 ml of complete medium. The adenoviral solution was replaced with complete medium, and cells were allowed to recover for 24 h before the experiment. Under these conditions, >95% of the cells are transduced (not shown).

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Cell Proliferation—INS832/13 cell proliferation was evaluated using a BrdUrd3 enzyme-linked immunoassay kit (Roche Applied Science). Adenovirus-transduced INS832/13 cells were seeded in 96-well plates (8 x 104 cells/well) and allowed to recover for 24 h. BrdUrd was added to the culture medium for 1 h, and cells were fixed, incubated with peroxidase-conjugated anti-BrdUrd antibody, and the immune complexes were quantified by enzyme-linked immunosorbent assay (Bio-Rad).

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Cell Death and Apoptosis桟ells were plated in 6-well plates and cultured until 80% confluent. Following transduction with -galactosidase or Foxo1 or treatment with 25 ?FONT size=-2>M H2O2 for 1 h to induce apoptosis (31), we measured caspase-3 activation and cell toxicity using the CaspACE and CytoTox 96 cytotoxicity (Promega) assay systems, respectively. The CytoTox 96 assay measures the release of cytoplasmic enzymes in the medium upon loss of membrane integrity as a result of cytotoxicity. Simultaneously, cells were harvested to measure apoptosis. After a brief centrifugation at 450 x g, cell pellets were resuspended in lysis buffer, and caspase-3 activity was determined by adding assay buffer containing the DEVD-p-nitroanilide substrate.

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Insulin Secretion and Total Insulin Content Assays—70% confluent INS832/13 cells were seeded in 24-well plates 1 day before use. On the day of the experiment, cells were washed and incubated for 30 min in 2.8 mM glucose KRBH buffer before incubation for 30 min at different glucose concentrations (2.8 and 16 mM) or 35 mM KCl to induce cell depolarization. At the end of the incubation, culture medium was collected, centrifuged to remove cells, and assayed for rat/human insulin content by radioimmunoassay (Linco, St. Charles, MO). Total insulin content was measured after acid ethanol extraction (32).

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Glucose Metabolism—We measured glucose oxidation as 14CO2 production from [U-14C]glucose and glucose utilization by measuring 3H2O production from [5-3H]glucose (33). We carried out the two determinations simultaneously and terminated the reactions by the addition of 40% perchloric acid. Tritiated water was separated by chromatography on AG1X8 column (Bio-Rad). 14CO2 was captured by glass fiber filters previously soaked in 5% KOH.

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Chromatin Immunoprecipitation—Chromatin immunoprecipitation assay kits (Upstate%20Biotechnology">Upstate Biotechnology, Inc., Lake Placid, NY) were used as described (34). 106 INS823/13 cells were growth-arrested by serum deprivation overnight, fixed in 1% formaldehyde, washed, and resuspended in lysis buffer. Samples were sonicated to shear DNA to lengths of 200-1,000 bp, and the material was immunoprecipitated with anti-Foxo1 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). DNA was recovered and amplified by PCR using oligonucleotides flanking the assayed promoter regions (34).

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Quantitative Real-time PCR Analysis of Malic Enzyme Isoforms—Cells were extracted using the RNeasy Mini Kit (Qiagen) with RNase-free DNase (Qiagen) from 6-well plates that had been set aside for RNA and were not used for the substrate stimulation. RNA was reverse transcribed (Stratagene, Stratoscript RT) to cDNA on a PTC-100 Thermocycler (Bio-Rad). Forward and reverse sequence primers (KECK facility, Yale University) and fluorescent-labeled internal probes (Applied Biosystems, TaqMan) were designed specifically for each malic enzyme isoform and normalized to actin. Sequences for primers and probes used were: ME1, 5'-ATGGAGAAGGAAGGTTTATCAAAG-3' and 5'-GGCTTCTAGGTTCTTCATTTCTT-3', internal probe, FAM-GGGCGTGCTTCTCTCACAGAAGA-TAMRA; ME2, 5'-GGCTTTAGCTGTTATTCTCTGTGA-3' and 5'-TGAATATTAGCAAGTGATGGGTAAA-3, internal probe, FAM-CCCGACACATCAGTGACACCGTTTTAMRA; ME3, 5'-AGATAAGTTCGGAATAAATTGCCT-3' and 5'-CATCATTGAACATGCAGTATTTGT-3', internal probe, FAM-GACTTTGCCAATGCCAATGCCTTC-TAMRA; actin, 5'-CCAGATCATGTTTGAGACCTTC-3' and 5'-CATGAGGTAGTCTGTCAGGTCC-3', internal probe, VIC-AGCCATGTACGTAGCCATCCAGGCT-TAMRA. Quantitative real-time PCR (Bio-Rad) using Quantitect PCR reagent (Qiagen) was evaluated using the Opticon 2 Monitor analysis software version 2.02 provided by MJ Research. Reaction efficiencies were determined to be greater than 98.7% for actin, ME1, ME2, and ME3.

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Cytosolic NADP+-dependent Malic Enzyme 1 Activity朏resh INS-1 cell extracts from the malic enzyme isoform knock-down studies were analyzed for malic enzyme activity. NADP+-dependent malic enzyme was measured spectrophotometrically (340 nm) at 32 癈 in a 96-well plate (250 祃/well). The assay was performed under the following reaction conditions: pH 7.4, 100 mM Tris/HCl, 1.0 mM MnCl2, 1.0 mM NH4Cl, 100 mM KCl, 1.25 mM NADP (freshly prepared), and 10 mM L-malate. All cell samples were run in duplicate with or without malate as substrate for test and control conditions, respectively. Cell extracts were added to all of the wells last, immediately shaken, and the absorbance read every 1 min for 40 min. Enzyme activity was determined by subtracting the activity of the control wells for each sample from the test wells run with L-malate. The resulting slopes of absorbance versus time were averaged and normalized to protein content.

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Mitochondrial NAD+ Preferred Malic Enzyme 2 Activity–Mitochondria were isolated by a modification of methods previously described (27). Briefly, INS-1 cells were grown to 80–100% confluence on 10 150-cm2 plates in RPMI. The cells were immediately placed on ice, washed with ice-cold phosphate-buffered saline and quickly extracted by the cell scraping technique in ice-cold isotonic mitochondria extraction buffer containing 65 mM sucrose, 215 mMD-mannitol, 5 mM HEPES, 3 mM MgCl2, 5 mM KH2PO4, and 5 mM KHCO3 (made fresh daily). The remainder of the protocol was carried out at 4 °C. Cell suspensions were centrifuged at low speed (100 x g) to gently pellet the cells. The supernatant was aspirated and the cell pellet was re-suspended in 1.0 ml of mitochondria buffer. The sample was transferred to a pre-cooled B-pestle Dounce and was passed 50 times, centrifuged for 3.5 min at 1.8 x 103 x g to remove heavy membranes, and re-extracted in isotonic buffer. The supernatants from both low speed spins were then centrifuged for 5 min at 8.5 x 103 x g to pellet out the mitochondria. The high-speed pellet was washed by re-suspension in mitochondria buffer and centrifuged a second time. The final pellet of isolated mitochondria was re-suspended in 1.0 ml of mitochondria buffer. Aliquots were taken for protein analysis (Bio-Rad, Lowry method) and the cytochrome c oxidase assay, the remainder of the sample was used for the 13C labeling studies. The cytochrome c oxidase assay was performed on intact and sonicated mitochondria to determine mitochondrial integrity. Mitochondrial integrity was determined to be greater than 79 ± 6%. Cytosolic contamination of less than 3% was determined by glycerol-3-P dehydrogenase activity in 300 mM triethanolamine-HCl buffer (pH 7.4, 0.13 mM NAD+). The freshly isolated mitochondria (1 mg protein) were then incubated in mitochondrial buffer with ADP (1.25 mM) in the presence of either [U-13C]fumarate (1 mM), or [U-13C]glutamate (1 mM) plus leucine (10 mM). After a 20-min incubation, the reaction was quenched with ice-cold perchloric acid (30%) and sonicated to disrupt the mitochondria. The solution was neutralized with potassium hydroxide, centrifuged, and the supernatant was lyophilized and resuspended in D2O for NMR analysis followed by LC/MS/MS determination of pyruvate isotopic enrichment.

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LC/MS/MS Analysis of [13C]Pyruvate桻uantitative analysis of the isotopomer distribution of [13C]pyruvate was performed with Applied Biosystems 4000 Q Trap LC/MS/MS system. Multiple reaction monitoring (negative-mode) of negatively charged ions from the TurboIonSpray probe was used to determine abundance of the unlabeled (86.9/43.3), single-labeled (87.9/43.3 and 87.9/44.3), double-labeled (88.9/45.3 and 88.9/44.3), and triple-labeled (89.9/45.3) pyruvate mass isotopomers. A 5-祃 sample of the mitochondrial extract was loaded onto a LUNA 5u C8 (2) 100A 250 x 4.6-mm column (Phenomenex, Torrance, CA) and eluted using a linear gradient starting with 75% buffer A, 25% B changing to 5% buffer A, 95% buffer B over 10 min. Buffer A was 95% water with 5% acetonitrile (2 mM ammonium acetate) and buffer B was 5% water with 95% acetonitrile (2 mM ammonium acetate). These conditions provided for the necessary baseline resolution of pyruvate from oxaloacetate because they ionize to produce similar ion fragment spectra. Pyruvate peak identification was based on the retention time of 13C-labeled and unlabeled standards.

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Insulin Secretion Analysis—Media extracts were measured for insulin concentration using the High Range Insulin ELISA kit (ALPCO) and analyzed on a spectrophotometric plate reader at 450 nm. All media extracts were normalized to cell protein concentration using a Micro-BCA Protein Assay Kit (Pierce).

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FIGURE 6.Summary of cytosolic and mitochondrial malic enzyme in INS-1 cells. In INS-1 cells, two pyruvate cycles differentially control insulin secretion. ME1 catalyzes a cytosolic pyruvate cycle that is necessary for the redox control of insulin secretion by generation of NADPH. Under glucose limiting conditions, ME2 couples anaplerotic metabolism of glutamate with insulin secretion by generating sufficient pyruvate for ATP synthesis and for export of malate for ME1 generation of NADPH.