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Dietary Iron Controls Circadian Hepatic Glucose Metabolism Through Heme Synthesis

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Dietary Iron Controls Circadian Hepatic Glucose Metabolism Through Heme Synthesis 1108 Diabetes Volume 64, April 2015 Judith A. Simcox,1 Thomas Creighton Mitchell,2 Yan Gao,1 Steven F. Just,2 Robert Cooksey,3 James Cox,1 Richard Ajioka,1 Deborah Jones,2 Soh-hyun Lee,2 Daniel King,1 Jingyu Huang,1and Donald A. McClain1,2,3 Dietary Iron Controls Circadian Hepatic Glucose Metabolism Through Heme Synthesis Diabetes 2015;64:1108–1119 | DOI: 10.2337/db14-0646 The circadian rhythm of the liver maintains glucose peripheral clocks also entrain to non-SCN zeitgebers. No- homeostasis, and disruption of this rhythm is associated tably, the liver is entrained to food intake and can become with type 2 diabetes. Feeding is one factor that sets the dyssynchronous from the SCN (2). Both circulating glu- circadian clock in peripheral tissues, but relatively little is cose and hepatic gluconeogenesis are regulated by circa- fi known about the role of speci c dietary components in dian factors, leading to the hypothesis that dyssynchrony that regard. We assessed the effects of dietary iron on between central and peripheral clocks may contribute to circadian gluconeogenesis. Dietary iron affects circadian the observed association between metabolic dysregulation glucose metabolism through heme-mediated regulation of the interaction of nuclear receptor subfamily 1 group d and altered sleep rhythms (3). fi member 1 (Rev-Erba) with its cosuppressor nuclear re- The precise contribution of speci c nutrients to setting ceptor corepressor 1 (NCOR). Loss of regulated heme the hepatic clock is incompletely understood. One micro- synthesis was achieved by aminolevulinic acid (ALA) treat- nutrient, iron, has significant effects on metabolism. Iron ment of mice or cultured cells to bypass the rate-limiting affects metabolic regulation through multiple mechanisms enzyme in hepatic heme synthesis, ALA synthase 1 including effects on AMP-dependent kinase, fuel preference, METABOLISM (ALAS1). ALA treatment abolishes differences in hepatic insulinsecretion,andregulation of the insulin-sensitizing glucose production and in the expression of gluconeo- adipokine adiponectin (3,4). Iron overload and excess dietary genic enzymes seen with variation of dietary iron. The iron are also significant risk factors for diabetes (5–7). Iron differences among diets are also lost with inhibition of is a particularly attractive candidate for contributing to heme synthesis with isonicotinylhydrazine. Dietary iron changes in circadian metabolism: not only is iron an essen- modulates levels of peroxisome proliferator–activated re- tial component of several proteins involved with electron ceptor g coactivator 1a (PGC-1a), a transcriptional acti- transport and metabolism, but also several circadian tran- vator of ALAS1, to affect hepatic heme. Treatment of mice with the antioxidant N-acetylcysteine diminishes PGC-1a scription factors bind heme. Heme, for example, is necessary variation observed among the iron diets, suggesting that for the formation of the complex of nuclear receptor sub- iron is acting through reactive oxygen species signaling. family1groupdmember1(Rev-Erba) with nuclear receptor corepressor 1 (NCOR), part of the negative arm of the cir- Circadian rhythms are endogenous cycles of behavioral cadian transcriptional feedback loop (8,9). Heme has also and physiological processes that are set by external signals been reported to bind the circadian proteins Clock and called zeitgebers. Light is the zeitgeber for the central clock Per2, although the functional relevance of this binding is of the suprachiasmatic nucleus (SCN) that controls circa- unknown and has been questioned (10–12). dian movement, feeding behavior, and thermoregulation To test the hypothesis that dietary iron intake may (1). These SCN-driven physiological factors contribute to affect circadian metabolic rhythms, we fed mice chow with the synchronization of clocks in peripheral tissues, but various iron concentrations, creating tissue iron levels 1Department of Biochemistry, University of Utah, Salt Lake City, UT This article contains Supplementary Data online at http://diabetes 2Department of Internal Medicine, University of Utah, Salt Lake City, UT .diabetesjournals.org/lookup/suppl/doi:10.2337/db14-0646/-/DC1. 3 Veterans Administration Research Service, VA Salt Lake City Health Care System, © 2015 by the American Diabetes Association. Readers may use this article as Salt Lake City, UT long as the work is properly cited, the use is educational and not for profit, and Corresponding author: Donald A. McClain, [email protected]. the work is not altered. Received 7 May 2014 and accepted 6 October 2014. See accompanying article, p. 1091. diabetes.diabetesjournals.org Simcox and Associates 1109 within the range found from normal variation in human Cell Culture diets. We demonstrate that dietary iron content affects HepG2 cells (American Type Culture Collection) were gluconeogenesis and circadian rhythm through modifying maintained in DMEM/Nutrient Mixture F-12 supplemented hepatic heme levels with concordant changes in Rev-Erba with 10% FBS and 0.1% primocin. The cells were plated binding to NCOR. Iron elicits these effects through perox- on six-well plates overnight and then treated with ferric isome proliferator–activated receptor g coactivator 1a ammonium citrate (FAC; 10 mmol), ALA (100 mg/mL), INH (PGC-1a)–mediated regulation of the rate-limiting enzyme (5 mmol), or a PBS no-treatment control for 12 h. After of heme synthesis in nonerythroid cells aminolevulinic acid the drug or iron treatment, the plate was synchronized synthase 1 (ALAS1). with a 100 nmol dexamethasone shock for 1 h; after this time, the media was removed, the cells washed with PBS, RESEARCH DESIGN AND METHODS and DMEM/Nutrient Mixture F-12 with treatment was Animal Studies replaced. Cells were harvested at 4-h intervals from the Three-month-old male C57BL/6J mice were fed diets of time of shock. Knockdown of PGC-1a was accomplished 35 mg/kg (TD.10211), 500 mg/kg (TD.10212), or 2 g/kg by PGC-1a small interfering RNA (siRNA) from Santa carbonyl iron (TD.10324) (Harlan Teklad, Madison, WI; Cruz Biotechnology (sc-38885), following the company’s by weight, 17.7% protein, 60.1% carbohydrate, and 7.2% general protocol with control siRNA (sc-37007) and trans- fat) for 6 weeks before in vivo physiological testing and fection reagent (sc-29528). 9 weeks before sacrifice. The 35-mg/kg diet is derived from RT-PCR the AIN93G diet, the 500-mg/kg diet is based on animal RNA was isolated from liver samples using RNeasy (Qiagen) facility diets that range from 200 to 500 mg/kg, and the and measured by Nanodrop (EPOCH). cDNA was synthe- 2-g/kg diet was selected due to the modest twofold increase sized with the SuperScript III First-Strand Synthesis System in liver iron stores, which is within the fourfold range seen (Invitrogen). Measurement of relative abundance was per- in hepatic iron among humans without iron-related pathol- formed by real-time PCR analysis using the Power SYBR ogy (13). The effects of heme iron were also assessed; heme Green Master Mix (Applied Biosciences) on a QuantStudio iron diet was provided to the mice as previously described 12 K Flex System with 384-well block. The standard curve by Ijssennagger et al. (14). was prepared by serial dilution of pooled cDNA from each The animals were maintained on a 12-h light/dark sample using five log standards. Rpl13 and cyB were se- cycle. Treatment of mice with 2 mg/mL aminolevulinic 10 lected as reference genes because they do not fluctuate tem- acid (ALA) (15) (#A167; Frontier Scientific), 1 mg/mL porally or vary with dietary iron (data not shown). Quantities isonicotinylhydrazine (INH) (16) (#I3377; Sigma-Aldrich), were calculated by QuantStudio expression suite software or 6.5 mg/mL N-acetylcysteine (NAC) (17) (#A9165; Sigma- based on the standard curve. Primers were designed using Aldrich) in their drinking water and adjusted to pH 7. the National Institutes of Health–facilitated Mouse Primer Water intake was monitored to ensure there were no Depot (http://mouseprimerdepot.nci.nih.gov/). variations. ALA treatment began after 6 weeks on diet and continued for 3 weeks concurrent with continued Western Blotting diet. Mice were treated with NAC or INH for 10 days after Antibodies used included Rev-Erba (Santa Cruz Biotech- 8 weeks on diet. In the case of NAC treatment, water was nology), NCOR, b-actin (Cell Signaling Technology), and changedevery2daystopreventoxidation.Priortoglucose PGC-1a (Abcam). The Criterion running and transfer sys- tolerancetest(GTT),micewerefastedfor6handthen tem with precast gels was used (Bio-Rad). challenged with 1 mg glucose/g body weight through i.p. Heme Measurement – injection. For pyruvate tolerance test (PTT), ad libitum fed Heme was measured by pyridine hemochrome spectro- micewerechallengedwith2mgpyruvate/gbodyweight photometric assay and high-performance liquid chroma- through i.p. injection. To monitor movement, food intake, tography (HPLC) (18). In the pyridine hemochrome assay, andrespiration,micewerehousedintheComprehensive heme was extracted from tissue samples with 500 mmol Laboratory Animal Monitoring system for 7 days with the NaOH and pyridine added to 40%. Wave scans measured fi rst 3 days as an acclimatization period (Comprehensive Lab from 450 to 620 nm using an Ultrospec 3000 spectropho- Animal Monitoring System; Columbus Instruments, Colum- tometer, after addition of 1 mol potassium ferricyanide bus, OH). For transcriptional studies, mice were harvested at and sodium dithionite crystals. Data were analyzed using 4-h time points at zeitgeber time (ZT) 2, ZT6, ZT10, ZT14, published extinction coefficients. For reverse-phase HPLC ZT18, and ZT22. Procedures were approved by the Institu- measurements, liver samples were homogenized in acidi- tional Animal Care and Use Committee of the University of fied acetone, filtered (0.45 mmol), and HPLC measure- Utah and the Veterans Administration. ments performed on a Waters 2690 pump with C18 Blood and Serum Measurements reverse-phase column (39 3 300 mm Waters mBondapak) Hematocrit and hemoglobin were measured by Vetscan hm5, and C18 precolumn (Waters 2690; Waters 996 phospho- whileinsulinandglucagonweremeasuredbyELISA(EMD diode array detector).
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