Flaxseed Lignan Increased Glucose Uptake by Human Red Blood Cells

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1 The Open Nutrceuticls Journl, 29, 2, Open Access Flxseed Lignn Incresed Glucose Uptke y Humn Red Blood Cells Yeong Rhee * nd Ardith Brunt 351 EML, NDSU Dept. # 262, PO Box 65, North Dkot Stte University, Frgo, ND, ; Deprtment of Helth, Nutrition nd Exercise Sciences, North Dkot Stte University, Frgo, North Dkot Astrct: The ojective of this study ws to determine if flxseed lignn (secoisolriciresinol diglucoside, SDG) would improve glucose uptke y humn red lood cells (RBCs). To mesure glucose uptke of RBCs, the RBCs were pretreted, in vitro, with SDG (, 1, 5, or 1 M) nd then were incuted with or without insulin (1 U/ml). After incution, 3-O-( 3 H-Methyl)-D-glucose uptke y the RBCs ws mesured. RBCs pretreted with 5 or 1 M SDG showed n increse in glucose uptke compred to control ( M SDG) or RBCs pretreted with 1 M SDG (p<.1). In ddition, glucose uptke ws incresed in RBCs with insulin stimultion compred to RBCs without insulin stimultion (p<.1). An interction etween SDG nd insulin ws lso oserved (p<.1). Pretretment of RBCs with SDG (5 or 1 μm) nd insulin incresed glucose uptke compred to those without insulin stimultion (p<.1). The results indicte potentil eneficil effects of SDG on glycemic control y incresing glucose uptke of RBCs. Key Words: Glucose uptke, flxseed lignn, SDG, ntioxidnt. INTRODUCTION Mintining glucose homeostsis is importnt in chronic disese prevention. Mny studies hve found tht hyperglycemi increses rective oxygen species (ROS), thus leding to incresed oxidtive stress nd oxidtive dmge s indicted y lipid peroxidtion or oxidtive DNA dmge in humns nd nimls [1-4]. Incresed oxidtive dmge to the cells my led to chronic disese development such s dietes nd crdiovsculr diseses [5, 6]. Glucose trnsport into cells is one of the mjor mechnisms of glucose homeostsis regultion. Humn erythrocytes re redily ccessile compred to other tissues, so these cells re used in studying glucose homeostsis [7-9]. Erythrocytes re lso good cell model for studying oxidtive stress induced dmge [4]. Antioxidnt pretretment of erythrocytes decresed oxidtive dmge y reducing lipid peroxidtion of the memrne [1]. In ddition, ntioxidnt pretretment protected red lood cells (RBCs) from structurl nd memrne lipid composition chnges which cused y oxidtive stress. The protection of RBCs from structurl nd memrne lipid composition chnges mintins the integrity of glucose trnsporter on RBC memrne, thus mintining norml glucose uptke y RBCs nd contriuting to glucose homeostsis [7-9]. Antioxidnts such s resvertrol, lph-lipoic cid, or lph-tocopherol supplementtion or pretretment of cells with n ntioxidnt significntly decresed lood glucose concentrtion nd incresed glucose uptke y erythrocytes, heptocytes, dipocytes, nd skeletl muscles in nimls *Address correspondence to this uthor t the 351 EML, NDSU Dept. # 262, PO Box 65, North Dkot Stte University, Frgo, ND, ; Deprtment of Helth, Nutrition nd Exercise Sciences, North Dkot Stte University, Frgo, North Dkot; Tel: ; Fx: ; E-mil: yeong.rhee@ndsu.edu [11-15]. Secoisolriciresinol diglucoside (SDG), known ntioxidnt, is found in flxseed [16-19]. Mny studies hve found the eneficil effects of SDG supplementtion on glycemic control in nimls due to decresed oxidtive stress nd incresed ntioxidnt sttus [6, 16, 18]. SDG inhiited in vitro DNA scissions nd linoleic cid peroxidtion which indicte ntioxidnt ctivity of SDG [2]. SDG supplementtion ws ssocited with decresed serum or pncretic mlondildehyde (MDA) nd ROS in white lood cells, which re used s indictors of oxidtive stress in the ody [6, 18]. Comprisons of ntioxidnt ctivity of SDG to tht of vitmin E showed etter ntioxidnt ctivities in SDG [19]. In our l, hypoglycemic effect of flxseed supplementtion ws found in impired glucose tolernt people [21]. Twelve weeks of flxseed supplementtion decresed verge fsting serum glucose concentrtions y 16% while whet rn supplementtion did not chnge fsting serum glucose concentrtions [21]. The current study ws conducted s the first step to determine the mechnism of the hypoglycemic effects of flxseed. Since the glucose lowering effects in the previous study were ttriuted to the ntioxidnt in flxseed, the current study used purified flxseed SDG. This study ws to determine if flxseed lignn, SDG, would improve in vitro glucose uptke y humn red lood cells. MATERIALS AND METHODOLOGY The reserch protocol ws pproved y the Institutionl Review Bord t North Dkot Stte University. Regents Purified flxseed SDG ws from Chromdex Co. (Snt An, CA); Solvle TM ws from PerkinElmer (Shelton, CT); 3% hydrogen peroxide ws from VWR (Btvi, IL); scintilltion cocktil ws from Reserch Product Interntionl /9 29 Benthm Open

2 82 The Open Nutrceuticls Journl, 29, Volume 2 Rhee nd Brunt Co. (Mount Prospect, IL); nd the reminders of the regents were from Sigm Co. (St. Louis, MO). RBC Isoltion Heprinized humn lood ws centrifuged to remove the plsm, nd then the red lood cell (RBC) pellet ws wshed with phosphte-uffered sline (PBS). Then the RBC pellet ws re-suspended t the originl lood volume with PBS. SDG Pretreted RBC SDG ws dissolved in.1% ethnol nd diluted into 1 M, 5 M, or 1 M. One ml of 1 M, 5 M, or 1 M SDG or.1% ethnol solution ( M SDG, control) ws dded to 2 ml of the RBC suspension, nd incuted in drk nd seled system t 37 C for 1 hour. After incution, the RBCs were wshed with PBS nd then re-suspended in PBS t the originl lood volume. RBC Incution with Insulin Porcine insulin ws dissolved in 1 M HCl nd then diluted into 1 μu/ml with PBS. One ml of 1 μu/ml insulin or 1M HCl/PBS (control) ws dded to 1 ml of SDG pretreted RBC suspension, nd incuted t 37 C for 3 minutes. After incution, the RBCs were wshed with PBS, nd re-suspended in PBS t the originl lood volume. For the glucose uptke ssy, 1 l of RBC suspension ws trnsferred into individul tues. Preprtion of Rdioleled Solution Five ml of.5 Ci/ml 3-O-( 3 H-Methyl)-D-glucose ws mixed with 5 ml of.5 mmol/l 3-O-Methyl-D-glucopyrnose (3OMG). Stop Solution Twenty mol/l mercuric chloride nd 1 mol/l phloretin were dded into 1 ml of ice-cold PBS immeditely efore use. RBC Glucose Uptke RBC glucose uptke ws mesured using modified method developed y Klepper et l. [22]. Glucose uptke ws initited y dding 2 l of 3 H-leled 3OMG solution into ech 1 l of RBC suspension which prepred t previous steps. Glucose uptke ws stopped y dding 1 ml of ice-cold stop solution t 3 sec, 1 min, nd 25 min. For the control, 1 ml of stop solution ws dded to 1 l of RBC suspension, nd then 2 l of 3 H-leled 3OMG solution ws dded. The RBC suspension ws centrifuged t 2, g for 5 min nd then wshed twice with 1 ml stop solution. To digest RBC pellet, 25 l of Solvle TM ws dded, incuted t 6 C for 1 hr, nd then cooled t room temperture. The RBC pellet ws further digested y dding 25 l of 3% hydrogen peroxide nd incuted t 6 C for 3 minutes. Finlly, 4 ml of scintilltion cocktil ws dded to ech vil nd then counted with scintilltion counter (Beckmn Coulter, Inc., Fullerton, CA) for 5 min count per smple. Dt Anlysis RBC glucose uptke dt were nlyzed with the SAS System (SAS 9.1; SAS Inst., Cry, NC) using n ANOVA model. The originl nlysis used n effects model specifiction which included ll min effects nd two- nd threefctor interctions for the primry fctors of interest. A Type I error rte (lph level) of.5 ws used for ll tests. Follow-up comprisons of mens were performed using cell mens model pproch which llows compring mens from ny tretment comintions rther thn just min effects mens. The Tukey-Krmer method ws used to control the experimentwise error rte for these follow-up tests. Dt were reported s men ± stndrd error. RESULTS Hemocytometer counting of the RBC suspension showed n verge numer of 7.88 x 1 6 RBC/ l, nd lso showed >95% of cell viility. There ws no time effect on glucose uptke of RBCs. However, the pretretment of RBCs with either 5 μm or 1 μm SDG incresed glucose uptke compred to RBCs without or with 1 μm SDG pretretment (p<.1, Fig. 1). No difference ws oserved in glucose uptke etween the 5 μm nd 1 μm SDG groups. 4 3 M 1 M 5 M 1 M SDG Concentrtion Fig. (1). Effects of SDG on RBC glucose uptke. The dt shown re representtive of three independent experiments, with the men nd stndrd error of the men shown for ech tretment group. Since there were no time effects on RBC glucose uptke, the men of ll different times for ech tretment group ws used for dt presenttion. Vlues with different lower cse letters re significntly different (p<.1).

3 SDG Effects on Glucose Uptke The Open Nutrceuticls Journl, 29, Volume 2 83 RBCs incuted with 1 μu/ml insulin incresed glucose uptke compred to RBCs without insulin (p<.1, Fig. 2). Incresed glucose uptke followed y SDG (5 μm or 1 μm) tretment ws lso oserved in groups without insulin stimultion (p<.1, Fig. 3). An interction etween SDG nd insulin ws oserved. Pretretment of RBCs with SDG (5 μm or 1 μm) nd insulin incresed glucose uptke compred to those without insulin stimultion (p<.1, Fig. 3). When compring n increse of glucose uptke followed y 5 μm SDG or U/ml 1 U/ml Insulin Concentrtion Fig. (2). Effects of insulin on RBC glucose uptke. The dt shown re representtive of three independent experiments, with the men nd stndrd error of the men shown for ech tretment group. Since there were no time effects on RBC glucose uptke, the men of ll different times for ech tretment group ws used for dt presenttion. Vlues with different lower cse letters re significntly different (p<.1). μm SDG without insulin pretretment to tht of cells with 1 μu/ml insulin nd SDG ( μm or 1 μm) pretretment, similr increse of glucose uptke ws oserved (p<.1, Fig. 3). However, no interction etween time nd SDG or time nd insulin ws oserved. DISCUSSION AND CONCLUSION Generlly, the contriution of glucose uptke y erythrocyte to glucose homeostsis is rther smll nd my not e mjor mechnism of the lood glucose lowering effect. However, due to esy ccessiility, erythrocytes were used in this study to mesure sl glucose uptke s the first step in the identifiction of the mechnism of glucose lowering effects of flxseed. Since this study ws to mesure glucose uptke y RBCs, the ntioxidnt ctivity of SDG ws not mesured in the current study. However, twelve weeks of flxseed supplementtion decresed plsm MDA concentrtion in glucose intolernt people in our previous study (Unpulished result). This result indictes tht SDG in flxseed functioned s n ntioxidnt. Significnt improvement of RBC glucose uptke y SDG tretment is in greement of findings of other studies of ntioxidnts [11, 12]. These studies suggested tht the protective function of n ntioxidnt ginst oxidtive stress improved in vitro glucose uptke nd led to decresed insulin resistnce in nimls [11, 13, 23, 24]. Although SDG effects on oxidtive stress in RBCs were not determined in the current study, SDG my hve cted similrly s other ntioxidnts used in other studies [23, 24]. High lood glucose levels generte ROS in individuls who hve dietes or impired glucose tolernce [25-27]. It 4 c c c 3 M 1 M 5 M 1 M M 1 M 5 M 1 M No Insulin Pretretment 1 U/ml Insulin Pretretment SDG Concentrtion Fig. (3). Effects of SDG nd insulin on RBC glucose uptke. The dt shown re representtive of three independent experiments, with the men nd stndrd error of the men shown for ech tretment group. Since there were no time effects on RBC glucose uptke, the men of ll different times for ech tretment group ws used for dt presenttion. Vlues with different lower cse letters re significntly different (p<.1).

4 84 The Open Nutrceuticls Journl, 29, Volume 2 Rhee nd Brunt is theorized tht glucose in the red lood cell cultures hd similr effects on ROS genertion. As result, this generted ROS my hve cused lower glucose uptke y RBCs without SDG tretment. Moreover, incresed ROS my hve incresed memrne lipid peroxidtion which distured memrne integrity nd decresed glucose trnsporter ctivtion on red lood cells. Improved glucose uptke y RBCs with SDG pretretment my e explined y decresed oxidtive stress, since SDG functions s n ntioxidnt. As seen in other studies of ntioxidnts [9], SDG is n ntioxidnt which my hve decresed lipid peroxidtion nd oxidtive dmge on RBCs, thus mintining the integrity of the cell memrnes nd cellulr structures including glucose trnsporters. Therefore, decresed lipid peroxidtion my hve led to incresed glucose uptke y RBCs. Insulin regultes glycemic control y stimulting cellulr glucose uptke. RBCs with SDG tretment incresed glucose uptke to similr level s tht of insulin stimulted cells. This result indictes tht SDG incresed sl glucose uptke y RBCs nd lso SDG hs the sme or similr hypoglycemic effect s insulin. Insulin pretretment of RBCs should not hve ffected glucose uptke directly, since RBCs trnsport glucose vi non-insulin medited glucose trnsporter. However, the interctions etween insulin nd SDG suggest tht SDG potentites insulin ction leding to incresed glucose uptke y RBCs. The significnt increse in glucose uptke y RBCs with SDG tretment my explin the lood glucose lowering effects of flxseed in humns s seen in our previous clinicl study [21]. Although there is report on vilility of SDG metolites, enterodiol nd enterolctone, in lood fter SDG consumption [28], no studies mesured the lood concentrtion of SDG fter norml flxseed lignn consumption. Therefore, it would e difficult to extrpolte the mount of SDG in the RBC suspension into n mount of SDG needed for whole humn ody to exert similr improvement of glucose uptke. However, individuls with n insulin/orl mediction regimen for dietes control would enefit y consuming flxseed in ddition to insulin/orl mediction. SDG in flxseed would reduce the required mount of insulin or orl mediction. These results demonstrte the eneficil effects of SDG on in vitro glucose uptke y humn RBCs. However, further investigtion is needed to determine the effects of SDG on the mechnism of cellulr glucose uptke in humns. For exmple, the in vitro cellulr glucose uptke following SDG metolite, enterodiol nd/or enterolctone stimultion nd in vivo cellulr glucose uptke following SDG supplementtion need to e mesured to identify the mechnism of hypoglycemic effects of flxseed in humns. ACKNOWLEDGEMENTS The uthors thnk Curt Doetkott for iding in sttisticl nlyses. This study ws funded y Grnts from NDSU HD&E College, Swenson Endowment, nd North Dkot EPSCoR. The equipment ws funded y Grnt from the NRI of the USDA CSREES, grnt # No conflict of interest exists. Both uthors contriuted to the mnuscript writing. REFERENCES [1] Kdiisk MB, Glden BC, Bird DD, et l. Biomrkers of oxidtive stress study II: Are oxidtion products of lipids, proteins, nd DNA mrkers of CCL4 poisoning? Free Rdic Biol Med 25; 38: [2] Kdiisk MB, Glden BC, Bird DD, et l. Biomrkers of oxidtive stress study III. Effects of the nonsteroidl nti-inflmmtory gents indomethcin nd meclofenmic cid on mesurements of oxidtive products of lipids in CCL4 poisoning. Free Rdic Biol Med 25; 38: [3] Dincer Y, Akcy T, Aldemir Z, Ilkov H. Assessment of DNA se oxidtion nd glutthione level in ptients with type 2 dietes. Mutt Res 22; 55: [4] Song F, Ji W, Yo Y, et l. Oxidtive stress, ntioxidnt sttus nd DNA dmge in ptients with impired glucose regultion nd newly dignosed type 2 dietes. 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5 SDG Effects on Glucose Uptke The Open Nutrceuticls Journl, 29, Volume 2 85 [2] Kitts DD, Yun YV, Wijewickreme AN, Thompson LU. Antioxidnt ctivity of the flxseed lignn secoisolriciresinol diglycoside nd its mmmlin lignn metolites enterodiol nd enterolctone. Mol Cell Biochem 1999; 22: [21] Rhee Y, Brunt A. Flxseed supplementtion ws effective in lowering serum glucose nd tricylglycerol in glucose intolernt people. JAMA 26; 9: [22] Klepper J, Grci-Alvrez M, O'Driscoll KR, et l. Erythrocyte 3-O-methyl-D-glucose uptke ssy for dignosis of glucosetrnsporter-protein syndrome. J Clin L Anl 1999; 13: [23] Estrd DE, Ewrt HS, Tskiridis T, et l. Stimultion of glucose uptke y the nturl coenzyme lph-lipoic cid/thioctic cid: Prticiption of elements of the insulin signling pthwy. Dietes 1996; 45, [24] Yworsky K, Somwr R, Rmll T, Tritschler HJ, Klip A. Enggement of the insulin-sensitive pthwy in the stimultion of glucose trnsport y lph-lipoic cid in 3T3-L1 dipocytes. Dietologi ; 43: [25] Hunt JV, Smith CC, Wolff SP. Autoxidtive glycosyltion nd possile involvement of peroxides nd free rdicls in LDL modifiction y glucose. Dietes 199; 39: [26] Bynes JW. Role of oxidtive stress in development of complictions in dietes. Dietes 1991; 4: [27] Thompson K, Godin D. Micronutrients nd ntioxidnts in the progression of dietes. Nutr Res 1995; 15: [28] Kuijsten A, Arts IC, Vree TB, Hollmn PC. Phrmcokinetics of enterolignns in helthy men nd women consuming single dose of secoisolriciresinol diglucoside. J Nutr 25; 135: Received: Jnury 1, 29 Revised: Ferury 23, 29 Accepted: Mrch 2, 29 Rhee nd Brunt; Licensee Benthm Open. This is n open ccess rticle licensed under the terms of the Cretive Commons Attriution Non-Commercil License ( which permits unrestricted, non-commercil use, distriution nd reproduction in ny medium, provided the work is properly cited.