CaV1.2 and CaV1.3 voltage-gated L-type Ca2+ channels in rat white fat adipocytes

in Journal of Endocrinology

Correspondence should be addressed to P A Smith: Paul.a.smith@nottingham.ac.uk
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L-type channel antagonists are of therapeutic benefit in the treatment of hyperlipidaemia and insulin resistance. Our aim was to identify L-type voltage-gated Ca2+ channels in white fat adipocytes, and determine if they affect intracellular Ca2+, lipolysis and lipogenesis. We used a multidisciplinary approach of molecular biology, confocal microscopy, Ca2+ imaging and metabolic assays to explore this problem using adipocytes isolated from adult rat epididymal fat pads. CaV1.2, CaV1.3 and CaV1.1 alpha1, beta and alpha2delta subunits were detected at the gene expression level. The CaV1.2 and CaV1.3 alpha1 subunits were identified in the plasma membrane at the protein level. Confocal microscopy with fluorescent antibodies labelled CaV1.2 in the plasma membrane. Ca2+ imaging revealed that the intracellular Ca2+ concentration, [Ca2 +]i was reversibly decreased by removal of extracellular Ca2+, an effect mimicked by verapamil, nifedipine and Co2+, all blockers of L-type channels, whereas the Ca2+ channel agonist BAY-K8644 increased [Ca2+]i. The finding that the magnitude of these effects correlated with basal [Ca2+]i suggests that adipocyte [Ca2+]i is controlled by L-type Ca2+ channels that are constitutively active at the adipocyte depolarized membrane potential. Pharmacological manipulation of L-type channel activity modulated both basal and catecholamine-stimulated lipolysis but not insulin-induced glucose uptake or lipogenesis. We conclude that white adipocytes have constitutively active L-type Ca2+ channels which explains their sensitivity of lipolysis to Ca2+ channel modulators. Our data suggest CaV1.2 as a potential novel therapeutic target in the treatment of obesity.

 

      Society for Endocrinology

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  • AllenDOBeckRRR 1986 Role of calcium ion in hormone-stimulated lipolysis. Biochemical Pharmacology 767772. (https://doi.org/10.1016/0006-2952(86)90244-3)

    • Search Google Scholar
    • Export Citation
  • ArmstrongCMGillyWF 1992 Access resistance and space clamp problems associated with whole-cell patch clamping. Methods in Enzymology 100122. (https://doi.org/10.1016/0076-6879(92)07007-B)

    • Search Google Scholar
    • Export Citation
  • ArnerPBernardSSalehpourMPossnertGLieblJSteierPBuchholzBAErikssonMArnerEHaunerHet al. 2011 Dynamics of human adipose lipid turnover in health and metabolic disease. Nature 110113. (https://doi.org/10.1038/nature10426)

    • Search Google Scholar
    • Export Citation
  • ArrudaAPHotamisligilGS 2015 Calcium homeostasis and organelle function in the pathogenesis of obesity and diabetes. Cell Metabolism 381397. (https://doi.org/10.1016/J.CMET.2015.06.010)

    • Search Google Scholar
    • Export Citation
  • AvasthyNJeremyJYDandonaP 1988 The role of calcium in mediating phorbol ester- and insulin-stimulated adipocyte lipogenesis. Diabetes Research 9195.

    • Search Google Scholar
    • Export Citation
  • BaumbachJHummelPBickmeyerIKowalczykKMFrankMKnorrKHildebrandtARiedelDJäckleHKühnleinRP 2014 A Drosophila in vivo screen identifies store-operated calcium entry as a key regulator of adiposity. Cell Metabolism 331343. (https://doi.org/10.1016/j.cmet.2013.12.004)

    • Search Google Scholar
    • Export Citation
  • BavleyCCFischerDKRizzoBKRajadhyakshaAM 2017 Cav1.2 channels mediate persistent chronic stress-induced behavioral deficits that are associated with prefrontal cortex activation of the p25/Cdk5-glucocorticoid receptor pathway. Neurobiology of Stress 2737. (https://doi.org/10.1016/J.YNSTR.2017.02.004)

    • Search Google Scholar
    • Export Citation
  • BegumNSussmanKEDrazninB 1992 Calcium-induced inhibition of phosphoserine phosphatase in insulin target cells is mediated by the phosphorylation and activation of inhibitor 1. Journal of Biological Chemistry 59595963.

    • Search Google Scholar
    • Export Citation
  • BentleyDCPulbutrPChanSSmithPA 2014 Etiology of the membrane potential of rat white fat adipocytes. American Journal of Physiology: Endocrinology and Metabolism E161E175. (https://doi.org/10.1152/ajpendo.00446.2013)

    • Search Google Scholar
    • Export Citation
  • BlackmorePFAugertG 1989 Effect of hormones on cytosolic free calcium in adipocytes. Cell Calcium 561567. (https://doi.org/10.1016/0143-4160(89)90018-3)

    • Search Google Scholar
    • Export Citation
  • BleicherSJFarberLLewisAGoldnerMG 1966 Electrolyte-activated lipolysis in vitro: modifying effect of calcium. Metabolism: Clinical and Experimental 742748. (https://doi.org/10.1016/s0026-0495(66)80010-0)

    • Search Google Scholar
    • Export Citation
  • BuonaratiORHendersonPBMurphyGGHorneMCHellJW 2017 Proteolytic processing of the L-type Ca2+ channel alpha 11.2 subunit in neurons. F1000Research 1166. (https://doi.org/10.12688/f1000research.11808.2)

    • Search Google Scholar
    • Export Citation
  • CarmenGYVíctorSM 2006 Signalling mechanisms regulating lipolysis. Cellular Signalling 401408. (https://doi.org/10.1016/j.cellsig.2005.08.009)

    • Search Google Scholar
    • Export Citation
  • CignarellaA 1994 Antithrombotic activity of nicardipine in spontaneously hypertensive rats. Pharmacological Research 273280. (https://doi.org/10.1016/1043-6618(94)80109-6)

    • Search Google Scholar
    • Export Citation
  • ClausenTMartinBR 1977 The effect of insulin on the washout of [45Ca]calcium from adipocytes and soleus muscle of the rat. Biochemical Journal 251255. (https://doi.org/10.1042/bj1640251)

    • Search Google Scholar
    • Export Citation
  • CoombsJSCurtisDREcclesJC 1956 Time courses of motoneuronal responses. Nature 11681169. (https://doi.org/10.1038/1781168a0)

  • DolphinAC 2016 Voltage-gated calcium channels and their auxiliary subunits: physiology and pathophysiology and pharmacology. Journal of Physiology 53695390. (https://doi.org/10.1113/JP272262)

    • Search Google Scholar
    • Export Citation
  • DrazninBSussmanKKaoMLewisDShermanN 1987 The existence of an optimal range of cytosolic free calcium for insulin-stimulated glucose transport in rat adipocytes. Journal of Biological Chemistry 1438514388.

    • Search Google Scholar
    • Export Citation
  • DrazninBSussmanKEEckelRHKaoMYostTShermanNA 1988 Possible role of cytosolic free calcium concentrations in mediating insulin resistance of obesity and hyperinsulinemia. Journal of Clinical Investigation 18481852. (https://doi.org/10.1172/JCI113801)

    • Search Google Scholar
    • Export Citation
  • EfendićSAlmBLöwH 1970 Effects of Ca++ on lipolysis in human omental adipose tissue in vitro. Hormone and Metabolic Research 287291. (https://doi.org/10.1055/s-0028-1095061)

    • Search Google Scholar
    • Export Citation
  • El HachmaneMFOlofssonCS 2018 A mechanically activated TRPC1-like current in white adipocytes. Biochemical and Biophysical Research Communications 736742. (https://doi.org/10.1016/j.bbrc.2018.03.050)

    • Search Google Scholar
    • Export Citation
  • FagerbergLHallströmBMOksvoldPKampfCDjureinovicDOdebergJHabukaMTahmasebpoorSDanielssonAEdlundKet al. 2014 Analysis of the human tissue-specific expression by genome-wide integration of transcriptomics and antibody-based proteomics. Molecular and Cellular Proteomics 397406. (https://doi.org/10.1074/mcp.M113.035600)

    • Search Google Scholar
    • Export Citation
  • FainJNGokmen-PolarYBahouthSW 1997 Wortmannin converts insulin but not oxytocin from an antilipolytic to a lipolytic agent in the presence of forskolin. Metabolism 6266. (https://doi.org/10.1016/S0026-0495(97)90169-4)

    • Search Google Scholar
    • Export Citation
  • FinkTLundPPilgaardLRasmussenJGDurouxMZacharV 2008 Instability of standard PCR reference genes in adipose-derived stem cells during propagation, differentiation and hypoxic exposure. BMC Molecular Biology 98. (https://doi.org/10.1186/1471-2199-9-98)

    • Search Google Scholar
    • Export Citation
  • FleischmannBKMurrayRKKotlikoffMI 1994 Voltage window for sustained elevation of cytosolic calcium in smooth muscle cells. PNAS 1191411918. (https://doi.org/10.1073/pnas.91.25.11914)

    • Search Google Scholar
    • Export Citation
  • GaionRKrishnaG 1983 Cyclic nucleotides and lipolysis in rat fat cells. Interaction between calcium ionophore A23187 and FCCP, uncoupler of oxidative phosphorylation. Life Sciences 571576. (https://doi.org/10.1016/0024-3205(83)90201-1)

    • Search Google Scholar
    • Export Citation
  • GaurSYamaguchiHGoodmanHM 1996a Growth hormone regulates cytosolic free calcium in rat fat cells by maintaining L-type calcium channels. American Journal of Physiology C1478C1484. (https://doi.org/10.1152/ajpcell.1996.270.5.C1478)

    • Search Google Scholar
    • Export Citation
  • GaurSYamaguchiHGoodmanHM 1996b Growth hormone increases calcium uptake in rat fat cells by a mechanism dependent on protein kinase C. American Journal of Physiology C1485C1492. (https://doi.org/10.1152/ajpcell.1996.270.5.C1485)

    • Search Google Scholar
    • Export Citation
  • GaurSSchwartzYTaiLRFrickGPGoodmanHM 1998 Insulin produces a growth hormone-like increase in intracellular free calcium concentration in okadaic acid-treated adipocytes. Endocrinology 49534961. (https://doi.org/10.1210/endo.139.12.6387)

    • Search Google Scholar
    • Export Citation
  • GorzelniakKJankeJEngeliSSharmaAM 2001 Validation of endogenous controls for gene expression studies in human adipocytes and preadipocytes. Hormone and Metabolic Research 625627. (https://doi.org/10.1055/s-2001-17911)

    • Search Google Scholar
    • Export Citation
  • HardyRWLadensonJHHruskaKAJiwaAHMcDonaldJM 1992 The effects of extracellular calcium and epinephrine on cytosolic-free calcium in single rat adipocytes. Endocrinology 36943702. (https://doi.org/10.1210/endo.130.6.1597165)

    • Search Google Scholar
    • Export Citation
  • HennemanEMendellLM 2010 Functional organization of motoneuron pool and its inputs. In Comprehensive Physiology. Hoboken NJ USA: John Wiley & Sons. (https://doi.org/10.1002/cphy.cp010211)

    • Search Google Scholar
    • Export Citation
  • HvarfnerABergströmRLithellHMörlinCWideLLjunghallS 1988 Changes in calcium metabolic indices during long-term treatment of patients with essential hypertension. Clinical Science 543549. (https://doi.org/10.1042/cs0750543)

    • Search Google Scholar
    • Export Citation
  • IzawaTKoshimizuEKomabayashiTTsuboiM 1983 Effects of Ca2+ and calmodulin inhibitors on lipolysis induced by epinephrine, norepinephrine, caffeine and ACTH in rat epididymal adipose tissue. Nihon Seirigaku Zasshi: Journal of the Physiological Society of Japan 3644.

    • Search Google Scholar
    • Export Citation
  • KellyKLJudeTCorkeyBE 1989 Cytosolic free calcium in adipocytes. Journal of Biological Chemistry 264 1275412757.

  • Le ScouarnecSBhasinNVieyresCHundTJCunhaSRKovalOMarionneauCChenBWuYDemolombeSet al. 2008 Dysfunction in ankyrin-B-dependent ion channel and transporter targeting causes human sinus node disease. PNAS 1561715622. (https://doi.org/10.1073/pnas.0805500105)

    • Search Google Scholar
    • Export Citation
  • LeeSCPapponePA 1997 Membrane responses to extracellular ATP in rat isolated white adipocytes. Pflugers Archiv 422428. (https://doi.org/10.1007/s004240050416)

    • Search Google Scholar
    • Export Citation
  • LipscombeDHeltonTDXuW 2004 L-type calcium channels: the low down. Journal of Neurophysiology 26332641. (https://doi.org/10.1152/jn.00486.2004)

    • Search Google Scholar
    • Export Citation
  • LouisCVan Den DaelenCTinantGBourezSThoméJPDonnayILarondelleYDebierC 2014 Efficient in vitro adipocyte model of long-term lipolysis: a tool to study the behavior of lipophilic compounds. In Vitro Cellular and Developmental Biology: Animal 507518. (https://doi.org/10.1007/s11626-014-9733-6)

    • Search Google Scholar
    • Export Citation
  • MartinBRClausenTGliemanntJ 1975 Relationships between the exchange of calcium and phosphate in isolated fat-cells. Biochemical Journal 121129. (https://doi.org/10.1042/bj1520121)

    • Search Google Scholar
    • Export Citation
  • MiyauchiAHruskaKAGreenfieldEMDuncanRAlvarezJBarattoloRColucciSZambonin-ZalloneATeitelbaumSLTetiA 1990 Osteoclast cytosolic calcium, regulated by voltage-gated calcium channels and extracellular calcium, controls podosome assembly and bone resorption. Journal of Cell Biology 25432552. (https://doi.org/10.1083/JCB.111.6.2543)

    • Search Google Scholar
    • Export Citation
  • N’GouemoPAkinfiresoyeLRAllardJSLovingerDM 2015 Alcohol withdrawal-induced seizure susceptibility is associated with an upregulation of CaV1.3 channels in the rat inferior colliculus. International Journal of Neuropsychopharmacology pyu123. (https://doi.org/10.1093/ijnp/pyu123)

    • Search Google Scholar
    • Export Citation
  • NiZSmogorzewskiMMassrySG 1994 Effects of parathyroid hormone on cytosolic calcium of rat adipocytes. Endocrinology 18371844. (https://doi.org/10.1210/endo.135.5.7525254)

    • Search Google Scholar
    • Export Citation
  • NiZSmogorzewskiMMassrySG 1995 Elevated cytosolic calcium of adipocytes in chronic renal failure. Kidney International 16241629. (https://doi.org/10.1038/ki.1995.226)

    • Search Google Scholar
    • Export Citation
  • PepeSBogdanovKHallaqHSpurgeonHLeafALakattaE 1994 Omega 3 polyunsaturated fatty acid modulates dihydropyridine effects on L-type Ca2+ channels, cytosolic Ca2+, and contraction in adult rat cardiac myocytes. PNAS 88328836. (https://doi.org/10.1073/pnas.91.19.8832)

    • Search Google Scholar
    • Export Citation
  • PershadsinghHALeeLYSnowdowneKW 1989 Evidence for a sodium/calcium exchanger and voltage-dependent calcium channels in adipocytes. FEBS Letters 8992. (https://doi.org/10.1016/0014-5793(89)81169-X)

    • Search Google Scholar
    • Export Citation
  • PfafflMW 2001 A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Research e45. (https://doi.org/10.1093/nar/29.9.e45)

    • Search Google Scholar
    • Export Citation
  • RaifmanTKKumarPHaaseHKlussmannEDascalNWeissS 2017 Protein kinase C enhances plasma membrane expression of cardiac L-type calcium channel, CaV1.2. Channels 604615. (https://doi.org/10.1080/19336950.2017.1369636)

    • Search Google Scholar
    • Export Citation
  • Ramírez-PonceMPAcostaJBellidoJA 1990 Electrical activity in white adipose tissue of rat. Revista Espanola de Fisiologia 133138.

  • RuedenCTSchindelinJHinerMCDeZoniaBEWalterAEArenaETEliceiriKW 2017 ImageJ2: ImageJ for the next generation of scientific image data. BMC Bioinformatics 529. (https://doi.org/10.1186/s12859-017-1934-z)

    • Search Google Scholar
    • Export Citation
  • RumiantsevDOPiotrovskiiVKMetelitsaVISlastnikovaIDMartsevich YuSKokurinaEV 1989 Serum binding of nifedipine and verapamil in patients with ischaemic heart disease on monotherapy. British Journal of Clinical Pharmacology 357361. (https://doi.org/10.1111/j.1365-2125.1989.tb05438.x)

    • Search Google Scholar
    • Export Citation
  • SattarNGillJM 2014 Type 2 diabetes as a disease of ectopic fat? BMC Medicine 123. (https://doi.org/10.1186/s12916-014-0123-4)

  • SchimmelRJ 1978 Calcium antagonists and lipolysis in isolated rat epididymal adipocytes: effects of tetracaine, manganese, cobaltous and lanthanum ions and D600. Hormone and Metabolic Research 128134. (https://doi.org/10.1055/s-0028-1093458)

    • Search Google Scholar
    • Export Citation
  • SchroederAMuellerOStockerSSalowskyRLeiberMGassmannMLightfootSMenzelWGranzowMRaggT 2006 The RIN: an RNA integrity number for assigning integrity values to RNA measurements. BMC Molecular Biology 3. (https://doi.org/10.1186/1471-2199-7-3)

    • Search Google Scholar
    • Export Citation
  • SchullaVRenströmEFeilRFeilSFranklinIGjinovciAJingXJLauxDLundquistIMagnusonMAet al. 2003 Impaired insulin secretion and glucose tolerance in beta cell-selective Ca(v)1.2 Ca2+ channel null mice. EMBO Journal 38443854. (https://doi.org/10.1093/emboj/cdg389)

    • Search Google Scholar
    • Export Citation
  • SchwartzYGoodmanHMYamaguchiH 1991 Refractoriness to growth hormone is associated with increased intracellular calcium in rat adipocytes. PNAS 67906794. (https://doi.org/10.1073/PNAS.88.15.6790)

    • Search Google Scholar
    • Export Citation
  • SeydouxJMuzzinPMoinatMPralongWGirardierLGiacobinoJ-PP 1996 Adrenoceptor heterogeneity in human white adipocytes differentiated in culture as assessed by cytosolic free calcium measurements. Cellular Signalling 117122. (https://doi.org/10.1016/0898-6568(95)02035-7)

    • Search Google Scholar
    • Export Citation
  • ShiLKoMLKoGY-P 2017 Retinoschisin facilitates the function of L-type voltage-gated calcium channels. Frontiers in Cellular Neuroscience 232. (https://doi.org/10.3389/fncel.2017.00232)

    • Search Google Scholar
    • Export Citation
  • SilverNCotroneoEProctorGOsailanSPatersonKLCarpenterGH 2008 Selection of housekeeping genes for gene expression studies in the adult rat submandibular gland under normal, inflamed, atrophic and regenerative states. BMC Molecular Biology 64. (https://doi.org/10.1186/1471-2199-9-64)

    • Search Google Scholar
    • Export Citation
  • SomaMRGottoAMGhiselliG 1989 Rapid modulation of rat adipocyte lipoprotein lipase: effect of calcium, A23187 ionophore, and thrombin. Biochimica et Biophysica Acta 307314. (https://doi.org/10.1016/0005-2760(89)90237-3)

    • Search Google Scholar
    • Export Citation
  • WangZLiVChanGCKPhanTNudelmanASXiaZStormDR 2009 Adult type 3 adenylyl cyclase-deficient mice are obese. PLoS ONE e6979. (https://doi.org/10.1371/journal.pone.0006979)

    • Search Google Scholar
    • Export Citation
  • XuWLipscombeD 2001 Neuronal Ca(V)1.3alpha(1) L-type channels activate at relatively hyperpolarized membrane potentials and are incompletely inhibited by dihydropyridines. Journal of Neuroscience 59445951. (https://doi.org/21/16/5944)

    • Search Google Scholar
    • Export Citation
  • YueFChengYBreschiAVierstraJWuWRybaTSandstromRMaZDavisCPopeBDet al. 2014 A comparative encyclopedia of DNA elements in the mouse genome. Nature 355364. (https://doi.org/10.1038/nature13992)

    • Search Google Scholar
    • Export Citation
  • ZieglerRJobstWMinneHFaulhaberJD 1980 Calciotropic hormones and lipolysis of human adipose tissue: role of extracellular calcium as conditioning but not regulating factor. Endokrinologie 7788.

    • Search Google Scholar
    • Export Citation