Phytanic acid stimulates glucose uptake in a model of skeletal muscles, the primary porcine myotubes

Transcription

1 Che et l. Lipis in Helth n Disese 2013, 12:14 RESEARCH Open Access Phytnic ci stimultes glucose uptke in moel of skeletl muscles, the primry porcine myotues Brit N Che 1, Niels Oksjerg 1, Lrs I Hellgren 2, Jco H Nielsen 3 n Jette F Young 1 Astrct Bckgroun: Phytnic ci (PA) is chlorophyll metolite with potentils in regulting glucose metolism, s it is nturl lign of the peroxisome prolifertor-ctivte receptor (PPAR) tht is known to regulte heptic glucose homeostsis. This stuy ime to estlish primry porcine myotues s moel for mesuring glucose uptke n glycogen synthesis, n to exmine the impct of physiologicl mounts of PA on glucose uptke n glycogen synthesis either lone or in comintion with insulin. Methos: Porcine stellite cells were culture into ifferentite myotues n tritite 2-eoxyglucose (2-DOG) ws use to mesure glucose uptke, in reltion to PA n 2-DOG exposure times n lso in reltion to PA n insulin concentrtions. The MIXED proceure moel of SAS ws use for sttisticl nlysis of t. Results: PA increse glucose uptke y pproximtely 35%, n the presence of insulin further increse the uptke, ut this further increse in uptke ws non- itive n less pronounce t high insulin concentrtions. There ws no effect of PA lone on glycogen synthesis, while the insulin stimultion of glycogen ws increse y 20% in the presence of PA. PA neither stimulte glucose uptke nor glycogen synthesis in insulin-resistnt myotues generte y excess glucose exposure. Conclusions: Primry porcine myotues were estlishe s moel of skeletl muscles for mesuring glucose uptke n glycogen synthesis, n we showe tht PA cn ply role in stimulting glucose uptke t no or inequte insulin concentrtions. Keywors: Phytnic ci, Plmitic ci, Insulin, Glucose uptke, Glycogen synthesis, Viility, Primry porcine myotues, Excess glucose, Free ftty cis Bckgroun Due to its high concentrtion of sturte ftty cis, iry ft intke hs een ssocite with increse risk of criovsculr iseses n type 2 ietes in humns [1,2]. However, recent met-nlysis of t from prospective cohort-stuies shows n inverse ssocition etween intke of iry proucts n the incience of oth criovsculr iseses n type 2 ietes [3], n ville t o not inicte ny increse risk of highft iry proucts compre to low-ft iry proucts [4]. Similrly, stuies using the o-chine ftty cis Corresponence: JetteF.Young@grsci.k 1 Deprtment of Foo Science, Arhus University, Blichers Allé 20, Tjele 8830, Denmrk Full list of uthor informtion is ville t the en of the rticle C15:0 n C17:0 s vlite iomrkers of iry ft intke showe tht higher intke of iry ft ws ssocite with ecrese risk of myocril-infrction n evelopment of the metolic synrome [5-8]. These finings suggest the existence of milk-ft prox; espite the sturte profile of milk-ft, it oes not seem to increse metolic risks, ut might ctully e protective. Hence, further reserch is necessry to elucite this controversy n to ientify potentil components in iry ft tht might e responsile for the protective effect. Milk is very rich source of potentilly ioctive lipis such s short-chine ftty cis n conjugte linoleic ci (CLA) [9,10]. Another ftty ci of interest in milk is phytnic ci (PA); C20 sturte ftty ci with 2013 Che et l.; licensee BioMe Centrl Lt. This is n Open Access rticle istriute uner the terms of the Cretive Commons Attriution License ( which permits unrestricte use, istriution, n reprouction in ny meium, provie the originl work is properly cite.

2 Che et l. Lipis in Helth n Disese 2013, 12:14 Pge 2 of 12 four methyl-rnches, which is known to e nturl lign of the nucler receptors peroxisome prolifertorctivte receptors (PPAR) n retinoi-x- receptors (RXR) [11-13]. The PPARs re lipi sensors, which ct y regulting oy glucose n lipi homeostsis [14], n it hs een shown tht PA enhnces glucose uptke in rt heptocytes, possily in PPAR-epenent mnner, t lest t high concentrtions [15]. Bse on these oservtions, it ws suggeste tht PA might improve glucose- homeostsis, n therey protect ginst the evelopment of the metolic synrome [16]. Diry proucts or met from ruminnts re mjor sources of PA in the humn iet [17]. The concentrtion of PA in plsm from helthy humns vry etween 0.04 n 11.5 μm [18], n is strongly epenent on the intke of ruminnt proucts [19]. Skeletl muscle fiers re the mjor site for insulinregulte glucose uptke, ccounting for over 70% of glucose isposl fter mel [20]. Furthermore, excess glucose n free ftty ci exposure le to insulin resistnce in these muscle fiers [21,22]. Hence, effects of PA on skeletl muscle glucose metolism re expecte to hve n impct on totl glucose homeostsis t the orgnism level. The min im of this stuy ws therefore to estlish primry porcine myotues s moel for glucose uptke n glycogen synthesis, n to exmine whether physiologicl concentrtions of PA cn lter glucose uptke n the rte of glycogen synthesis in primry porcine myotue cultures, either lone or in comintion with insulin. Results Viility ssy Primry porcine myotues were incute with oses of PA rnging from μm (Tle 1). The viility of the myotues ws not ffecte y 1 10 μm PA. Exposure of the myotues to 20 μm PA mrkely reuce their viility y out 20%. This viility roppe y 36% fter exposure to 500 μm PA. For comprtive resons, the effect of plmitic ci (PAM) on viility ws lso ssye (Tle 1). Exposure of myotues to 1 or 10 μm PAM h no effect on viility while 50 μm PAM reuce their viility y out 15% n y 500 μm PAM the viility of the myotues were reuce to only 54%. Optimiztion in reltion to glucose uptke ssy Glucose uptke in primry porcine myotues ws insulin-ose-epenent (Figure 1). At 0.1 nm insulin, significnt increse in glucose uptke ws recore n t suprphysiologicl concentrtion of 500 nm insulin, 70% increse in uptke ws otine. To verify the presence of crrier-meite glucose uptke in the primry porcine myotue moel, ifferent oses of Tle 1 Effect of ftty cis on the viility of primry porcine myotues Ftty ci Fol chnge in viility concentrtion (μm) PA PAM Control (0) 1 ± ± ± ± ± ± c ± ± c ± ± c ± ± c ± ± Differentite porcine myotues were incute with ifferent concentrtions of ftty cis (PA or PAM) for 24 h, n then trete with WST-1 regent s mentione in the methos. Control smples contine experimentl mei without ftty cis. Dt re expresse s LSmen vlues from five-eight replicte wells, crrie out in three seprte experiments with cells isolte from ifferent pig ech time. LSmens with ifferent letters (-) within ech row enote significntly ifferent responses relte to ftty ci tretments; (P < 0.05). cytochlsin B (cyto B) (0 50 μm) were e to the ifferentite myotues (Figure 1). At 10 μm cyto B, glucose uptke in myotues with n without insulin ws reuce to 54 n 40%, respectively, when compre to tht of control cells without insulin or cyto B ition. In the presence of μm cyto B, the glucose uptke ws reuce to pproximtely 70-90% of controls without cyto B, irrespective of insulin ition. Glucose uptke s function of PA incution time n 2-DOG incution time Bse on the results from the viility stuies n the concentrtion profile of PA in humn plsm, glucose uptke ssys were performe in myotues tht were incute with 10 μm PA over perio of 24 h to efine n effective incution time with PA (Figure 2). No significnt increse in glucose uptke ws oserve fter 1 h incution with PA. There ws, however, tenency of elevte glucose uptke etween 4 8 h of incution with PA followe y temporry mrke ecrese etween h of incution, when compre to 1 h of incution. To estimte n optiml incution time for 2-DOG to chieve effective uptke, myotues were expose to 2- DOG for 5 60 min with or without 10 μm PA (Figure 2). There ws generl stey increse in glucose uptke with incresing time of incution with 2- DOG. The steepest increse in 2-DOG uptke ws oserve uring the first 15 min, while the process ws slowest uring the lst 10 min of incution. In the presence of PA, glucose uptke tene to e higher t ll the

3 Che et l. Lipis in Helth n Disese 2013, 12:14 Pge 3 of 12 Fol chnge in glucose uptke (pm) c c c c c Insulin concentrtion (nm) Fol chnge in glucose uptke (pm) cyto B concentrtion (µm) Figure 1 Glucose uptke in porcine myotues in response to insulin n cyto B. Glucose uptke ws performe on ifferentite porcine myotues in the presence of vrying concentrtions of insulin (), or vrying concentrtions of cyto B with (full circles) or without (open circles) 500 nm insulin (). Control smples lcke cyto B. Dt re expresse s LSmen vlues of four replicte wells, crrie out in t lest two seprte experiments with cells isolte from ifferent pig ech time. enotes LSmens with significnt ifference in responses with or without insulin; (P < 0.05). ifferent incution times, ut this ifference ws significnt only fter 60 min of incution with 2-DOG. Glucose uptke in response to PA n PAM When testing the effect of PA on glucose uptke (Tle 2), 23% increse in uptke ws note even t the suphysiologicl concentrtion of 0.5 μm PA. Incresing the PA concentrtion further i not significntly increse the uptke, lthough verge uptke increse to etween 30-35%. The effect of 1 10 μm PA coul not e mimicke y sme concentrtions of PAM (Tle 2). However, when myotues were expose to higher oses of PAM, there were mrke chnges in glucose uptke. Glucose uptke roppe to 75% when the myotues were expose to 100 μm PAMnt200μM n 400 μm PAM, the glucose uptke ility of the myotues ws only 50% n 30%, respectively, when compre to controls. Glucose uptke n glycogen synthesis with or without PA s function of insulin The impct of PA on glucose uptke n glycogen synthesis ws lso stuie in reltion to vrious insulin

4 Che et l. Lipis in Helth n Disese 2013, 12:14 Pge 4 of 12 Fol chnge in glucose uptke (pm) PA incution time (h) Fol chnge in glucose uptke (pm) c c DOG incution time (min) Figure 2 Glucose uptke with or without PA s function of 2-DOG incution time. Glucose uptke ws performe on ifferentite myotues tht h een incute with () 10μM PA for ifferent times n then trete with 2-DOG for 30 min, or () with (full circles) or without (open circles) 10 μm PA for 4 h n fterwrs trete with 2-DOG for 5 60 min. Dt re expresse s LSmen vlues of triplicte wells crrie out in three seprte experiments with cells isolte from ifferent pig ech time. LSmens with ifferent letters (-e) enote significntly ifferent responses relte to incution times, while enotes LSmens with significntly ifferent responses with or without PA; (P < 0.05). e concentrtions. Aition of 10 μm PA cuse further increse in glucose uptke t ll levels of insulin (Figure 3), ut significnt improvement ws only chieve t lower insulin concentrtions (0 n 0.1nM). The incorportion of glucose into glycogen increse in response to insulin tretment from nm, with significnt increse t 10 n 100 nm insulin (Figure 3). Overll, glycogen synthesis ws not significntly ffecte y PA lone, regrless of the increse tenency. However, in the presence of 10 nm insulin, PA cuse n itionl increse in glycogen synthesis of out 20%. Exposure of myotues to extrcellulr glucose The viility of the myotues fter exposure to excess glucose (12 mm) ws not significntly ffecte (Figure 4). The ition of 7, 10 n 15 mm of extrcellulr glucose reuce glucose uptke to out 80, 65 n 23%,

5 Che et l. Lipis in Helth n Disese 2013, 12:14 Pge 5 of 12 Tle 2 Glucose uptke in response to PA n PAM Ftty ci Glucose uptke concentrtion (μm) PA PAM Control (0) 0 ± ± ± ± ± ± ± ± ± ± ± ± c ± ± Glucose uptke ws crrie out on ifferentite myotues tht h een incute with 1 60 μm PA or 1n 10 μm PAM for 4 h. PAM from μm ws ministere for 24 h. Control smples lcke ftty cis. Dt re expresse s LSmen vlues of triplicte wells, crrie out in three seprte experiments with cells isolte from ifferent pig ech time. LSmens with ifferent letters (-) within ech row enote significntly ifferent responses relte to ftty ci tretments; (P < 0.05). respectively, when compre to myotues expose to only 4 mm of extrcellulr glucose (Figure 4). Higher mounts of extrcellulr glucose i not cuse further rops in glucose uptke. While 10 μm PA n 10 nm insulin cuse n pproximte 40 n 75% increse, respectively, in glucose uptke in myotues expose to 6 mm extrcellulr glucose, no effect of neither PA nor insulin ws oserve when myotues were expose to 14 mm extrcellulr glucose (Figure 4c). Glycogen synthesis ws reuce to 25% when myotues were trete with 14 mm glucose, compre to myotues trete with 6 mm glucose (Figure 4). The mrke effect of 10 nm insulin on glycogen synthesis in the presence of 6 mm glucose ws olishe y exposing the myotues to 14 mm glucose (Figure 4). Discussion Phytnic ci (PA) is nturl lign n ctivtor of the PPARs, specificlly PPAR-α n γ [23]. The PPARs hve een implicte not only in the regultion of ipose tissue evelopment n insulin signling [24] ut lso in moulting insulin sensitivity of muscles [25,26] n incresing exercise enurnce [27]. It hs een hypothesize tht PA cn function s nturl PPAR gonist n therefore, e useful in the prevention n tretment of ietes [28]. Hence, PA my hve positive influence on the regultion of glucose metolism. As skeletl muscle is the mjor site of insulin-epenent glucose uptke [20], we estlishe primry myotue moel to stuy the effect of PA on glucose-homeostsis. The porcine primry myotue moel shres mny similrities to the humn myocytes n hs fewer rtifcts when compre to immortl cell line systems [29]. Stellite cells were grown into myolsts (Figure 5 n c) n successfully ifferentite into chrcteristic multinuclete myotues (Figure 5 n ), just s in previous finings [30]. Initilly, the suitility of the primry porcine myotue moel ws teste, n ssy conitions optimize in reltion to oth glucose ssy compoun (2-DOG) n PA exposure. Viility ssys were performe to etermine n pproprite concentrtion of PA tht i not compromise the cells metolic ctivities. The viility of the myotues ws noticely reuce when expose to concentrtions of PA equl to or ove 20 μm (Tle 1). The toxic effect of PA t 20 μm or ove my e ttriute to poptosis of the cells, s shown previously y stuies in humn skin firolsts n rt liver [31,32]. The verge concentrtion of PA in humn plsm hs een reporte to e etween μm [18,19,33], with higher vlues foun preominntly in met eters n iry prouct consumers [18,19]. Bse on this, n the results from our viility stuies, the working concentrtion of PA ws chosen s 10 μm. Plmitic ci (PAM); ftty ci of sme chin length s PA ut without the rnching methyl-groups ws use s control, n we showe tht 1 n 10 μm PAM h no effect on myotue viility. At concentrtions of 50 μm PAM n ove, the viility of the myotues were reuce, possily in mnner similr to tht of higher mounts of PA [31,32]. The non-metolizle nlogue of glucose; 2- eoxyglucose (2-DOG), is suitle mrker for the mesurement of muscle glucose trnsport s it cn e trnsporte into cells just like glucose [34], n it ws use in this stuy to mesure glucose uptke. Glucose trnsport in muscles is insulin-epenent n meite y GLUT-4 trnsporters, s well s insulin-inepenent, meite y GLUT-1 trnsporters [35]. Insulin tretment of vrying concentrtions cuse ose-epenent increse in glucose uptke in the myotues (Figure 1), proving tht theprimryporcinemyotueshvefunctionlinsulinstimulte GLUT-4 trnsloction to the plsm memrne [36]. The ction of oth GLUT-1 n GLUT-4 in the crrier-meite glucose uptke ws checke y tretment of the myotues with cytochlsin B (cyto B); rug known to inhiit glucose trnsport y ining to glucose trnsporters with higher ffinity thn glucose [37,38]. From our oservtion, cyto B inhiite oth the insulin- n noninsulin-meite glucose uptke to similr mgnitue in primry porcine myotues (Figure 1). The reuction in glucoseuptkeycytobconfirmsthtoththeinsulinn the non-insulin-meite trnsport systems re functionl in our primry porcine myotue moel. Our oservtion with cyto B lso showe tht out 20% of

6 Che et l. Lipis in Helth n Disese 2013, 12:14 Pge 6 of 12 Fol chnge in glucose uptke(pm) c c Insulin concentrtion(nm) Fol chnge in glycogen synthesis (pm/mg protein) c Insulin concentrtion (nm) Figure 3 Glucose uptke n glycogen synthesis with or without PA s function of insulin. Glucose uptke () ws crrie out on ifferentite myotues tht h een incute with (full circles) or without (open circles) 10 μm PA for 4 h, followe y ifferent concentrtions of insulin uring the lst 1 h. Glycogen syntheses () were performe on myotues trete with (full circles) or without (open circles) 10 μm PA for 4 h, followe y nm insulin for 2 h. Control smples lcke PA n insulin. Dt re expresse s LSmen vlues collecte from three-four wells, crrie out in three seprte experiments with cells isolte from ifferent pig ech time. LSmens with ifferent letters (-) enote significntly ifferent responses relte to the insulin concentrtion, while enotes LSmens with significntly ifferent responses with or without PA; (P < 0.05). c glucose uptke in the myotues ws not ffecte y cyto B. One cn speculte tht miniml stey stte of nonspecific glucose uptke is mintine in the porcine cultures. It is lso likely tht other GLUT receptors thn GLUT1n4representinthemyotues[39],ncyto B cnnot in to them. In tht cse glucose trnsport is possile even in the presence of cyto B. A iphsic effect on glucose uptke ws oserve uring the 24 h incution of myotues with 10 μm PA (Figure 2). Since only sutle increse in glucose

7 Che et l. Lipis in Helth n Disese 2013, 12:14 Pge 7 of 12 c 2.5 Fol chnge in viility Fol chnge in glucose uptke (pm) control 10 µm PA 10 nm Ins Glucose concentrtion (mm) Glucose concentrtion (mm) Fol chnge in glucose uptke (pm) c Fol chnge in glycogen synthesis (pm/mg protein) control 10 µm PA 10 nm Ins Glucose concentrtion (mm) Glucose concentrtion (mm) Figure 4 Exposure of myotues to extrcellulr glucose. Differentite myotues trete with two ifferent concentrtions of glucose for 24 h were sujecte to viility ssy (). Glucose uptke ws lso performe on myotues expose to vrious glucose concentrtions (). Myotues expose to 6 mm glucose (controls) or 14 mm glucose for 24 h in the sence (open rs) or presence of PA (lck rs) or insulin (grey rs) were sujecte either to (c) glucose uptke or () glycogen synthesis mesurements. PA ws ministere for 24 h while insulin ws given uring the lst 1 h of incution for glucose uptke mesurements n 2 h for glycogen synthesis. Dt re expresse s LSmen vlues of four replicte wells, crrie out in three seprte experiments with cells isolte from ifferent pig ech time. LSmens with ifferent letters ( n ) enote significntly ifferent responses relte to PA or insulin tretments, while sttisticl ifference in LSmens etween controls n myotues given 14 mm glucose is enote with ; (P < 0.05). uptke ws notice fter 4 n 8 h exposure to PA, it is less likely tht the susequent fll in glucose uptke is ue to epletion of GLUT epots. Other unknown fctors coul, thus, e responsile for this rop in glucose uptke. A rise in glucose uptke t 24 h coul e linke to consequent up-regultion of trnscripts responsile for the trnsltion of proteins tht skew up glucose uptke. Glucose uptke hs een shown to e liner for over 20 min in humn skeletl muscle cell line stuy [40], n in the present stuy, the steepest increse in glucose uptke ws oserve uring the first 15 min of incution with 2-DOG n y 45 min, the curve strte to level off (Figure 2). It is possile tht fter 45 min incution with 2-DOG, prtil sturtion of the norml glucose pthwy is ttine. Following these oservtions, the incution time with 2-DOG ws set t 30 min. Consiering the effects of PA on glucose uptke, it is noteworthy tht concentrtion s low s 1 μm cuse 25% increse in glucose uptke. Incresing the PA concentrtion to 10 μm slightly increse glucose uptke further (Tle 2), while equivlent mounts of PAM h no effect on glucose uptke. The effect of PA ws, thus, specific effect, which is in greement with similr stuy in rt heptocytes, in which 100 μm PA mrkely increse glucose uptke following incution with 2-DOG, when compre to ocoshexenoic ci [15]. The reuction in glucose uptke oserve t PAM concentrtion of

8 Che et l. Lipis in Helth n Disese 2013, 12:14 Pge 8 of 12 c Figure 5 Microscopic view of proliferting n ifferentiting porcine stellite cells. Proliferting myolsts t 80% confluence () or myotues t y 2 of ifferentition () were stine s escrie in the methos to revel the single nuclei cells of myolsts (c) n multinuclei tuulr structure of myotues (). Picture mgnifiction ws 100 x. over 100 μm (Tle 2) ws prtly ue to inhiition of glucose trnsport ctivity [41], ut lso most likely consequence of stress [31]. The effect of PA iminishe with incresing insulin stimultion of glucose uptke (Figure 3). This coul inicte similr moe of ction etween insulin n PA. Our results lso inicte tht the PA concentrtions normlly foun in plsm (> 1 μm), re sufficient to enhnce non-insulin epenent glucose uptke n tht ltertion of plsm PA concentrtions within norml physiologicl rnge, only hve minor effects. Hence, we suggest tht norml levels of PA is relevnt fctor in controlling fsting glucose homeostsis in vivo n tht increse intke of PA only will ffect glucose uptke in sujects with extremely low PA-levels (for exmple strict vegetrins with no intke of iry proucts or ruminnt met). Despite the fct tht 10 μm PA inuce n increse glucose uptke in the sence of insulin (Figure 3), it i not enhnce the rte of glycogen synthesis (Figure 3). Thus, PA t physiologicl concentrtions increses glucose uptke in the sence of insulin, without cusing concomitnt increse in glycogen synthesis, why the sore glucose must e shunte into other metolic pthwys. However, the comintion of 10 μm PA n 10 nm insulin more thn oule the rte of glycogen synthesis, compre to the effect of insulin lone (45% vs. 20%). This cretes somewht proxicl sitution tht incresing insulin concentrtions ttenute the effect of PA on glucose uptke, ut enhnce its effect on glycogen synthesis. Hence, the two effects must e expline y ifferent mechnisms of ction of PA. It shoul e note, tht the effect of PA on oth glucose uptke n glycogen synthesis is not generl effect of ftty cis, s the sme concentrtion of PAM i not cuse these effects, neither with nor without insulin (t not shown). Previous stuies hve shown tht excess glucose cuses glucose toxicity tht is mnifeste y the inhiition of glucose uptke n inuction of insulin resistnce in the muscles [42]. The toxicity of excess glucose is ue to the ctivtion of the hexmine pthwy n prouction of glucosmine, which inhiits insulinstimulte glucose uptke n susequently glycogen synthesis [43]. Our oservtions tht incresing mounts of glucose inhiit glucose uptke (Figure 4 n c) n glycogen synthesis (Figure 4) re in greement with

9 Che et l. Lipis in Helth n Disese 2013, 12:14 Pge 9 of 12 these finings. Excess glucose i not ffect the viility of the myotues (Figure 4), ruling out the possiility tht the fll in glucose uptke ws ue to ecrese viility of the myotues. These finings stress the role of excess glucose in generting insulin resistnce; it is therefore lso noteworthy tht neither PA nor insulin h ny effect on glucose uptke or glycogen synthesis in myotues tht h een expose to excess glucose. Conclusion In the present stuy, we hve een le to generte suitle primry porcine cell moel for stuying oth insulin n non- insulin-stimulte glucose uptke t physiologicl levels of insulin, s 0.1 nm insulin inuces significnt increse in glucose uptke. We hve shown tht PA lone cn improve glucose uptke ut not glycogen synthesis n tht uring glucose uptke, PA proly competes with insulin in insulin-signling. PA cn neither stimulte glucose uptke nor glycogen synthesis in insulin-resistnt cells generte y exposure to excess glucose. A potentil role for PA my, thus, e stimulting glucose uptke in muscle cells t inequte insulin concentrtions, lthough the consequences of incresing glucose uptke without concomitnt increse in glycogen synthesis nee to e stuie further. Methos Mterils n regents Humn insulin, PA, PAM, imethyl sulfoxie (DMSO), cyto B, essentilly ftty ci-free ovine serum lumin (eftte BSA), glycogen, hemtoxylin, chlorl hyrte, cytosine rinosie n ntiiotics were ll purchse from Sigm. Dulecco s Moifie Egle s Meium (DMEM), fetl ovine serum (FBS), horse serum (HS) n phosphte uffere sline (PBS) were otine from Life Technology. The WST-1 regent ws otine from Boehringer Mnnheim. Mtrigel regent ws from Becton Dickinson, n the BCA kit ws from Pierce Rockfor. [1-3H] - 2-DOG ws purchse from GE Helthcre n [1-14 C]-D-glucose from PerkinElmer. Buffers n mei The prolifertion growth mei (PGM) consiste of DMEM contining 22 mm D-glucose, 10% FBS, 10% HS, 1% penicillin-streptomycin, % mphotericin B n 0.2% gentmycin. The first ifferentition mei (DM1) ws s PGM ut without HS n D-glucose ws reuce to 6 mm. The secon ifferentition mei (DM2) ws s DM1, except for reuction of FBS to 5% n ition of cytosine rinosie to stop prolifertion. The thir ifferentition mei (DM3) ws s DM2 ut lcke serum. Hepes-uffere sline (HBS) ph 7.4 consiste of 20 mm Hepes, 140 mm NCl, 5 mm KCl, 2.5 mm MgSO 4 n 1 mm CCl 2. The storge meium ws me up of PGM n 10% DMSO. Hemtoxylin solution (100 ml) consiste of 5 g hyrte potssium sulphte, 0.01 g soium iote, 0.1 g hemtoxylin ye, 5 g chlorl hyrte n 0.1 g cetic ci to improve shelf-life of the solution. The experimentl mei normlly consiste of the stock mei (2.5 mm D-glucose n eftte BSA) ut where inicte, inclue ifferent oses of insulin n/or glucose n ftty cis. Ftty cis were soluilize either in DMSO or ethnol. Ethnol ws evporte uner strem of liqui nitrogen while the finl mount of DMSO in the experimentl mei ws less thn 0.1%. The experimentl mei with ftty ci ws sterilize using sterile filter n left overnight t room temperture to ensure efficient ining of eftte BSA to the free ftty cis. In this stuy, the ftty ci: BSA rtio ws limite to 5:1. The mount of glucose, DMSO n eftte BSA in control smples ws equivlent to tht of trete smples, unless stte otherwise. Mei for glycogen nlysis consiste of 5 mm D-glucose, 0.2 μci [1-14 C]-D-glucose n 0.1% eftte BSA in HBS. Glycogen crrier stock solution consiste of 0.5 g glycogen in 50 ml 30% KOH solution. Isoltion n culture of primry porcine stellite cells Primry stellite cells were otine from the left semimemrnosus muscle of six weeks-ol femle pigs, weighing etween kg following metho escrie y Theil et l. [44]. Isolte cells were plce in storge meium n store in liqui nitrogen until use. Cells were thwe in 37 C wter th n seee on 4% mtrigel-cote pltes contining PGM. After 2 ys, cells were wshe with 1xPBS, ph 7.4 (with C 2+,Mg 2+ ) n llowe to proliferte for 3 4 ys to 80% confluence (Figure 5 n c). Cells were given DM1 until 100% confluent, n therefter DM2 ws given to stop prolifertion n initite ifferentition. At this stge, the cells were enote to e t their first y of ifferentition. Cells were given DM2 until y 2 of ifferentition, where fully evelope myotues were oserve microscopiclly (Figure 5 n ). Myotues were given DM3 16 h prior to experimentl tretment. When the experimentl tretment ws ministere for more thn 4 h, DM3 ws omitte. Cell culturing ws performe t 37 C with 5% CO 2 n t 100% humiity. Hemtoxylin stining Cells were rinse twice with 1xPBS (without C 2+,Mg 2+ ) n fixe with ice-col methnol for 5 min. The methnol ws replce y hemtoxylin solution for 10 min, fter

10 Che et l. Lipis in Helth n Disese 2013, 12:14 Pge 10 of 12 which the ye ws spirte n cells were rinse severl times to iscr unwnte stin. Cells were viewe for pproprite stining on microscope t 100 mgnifiction. Viility test After cell growth, ifferentition n incution with experimentl mei for 24 h, myotues were rinse with 1xPBS (with C 2+, Mg 2+ ) n WST-1 regent wsusesescrieyokuret l. [45]. The genertion of formzn ye y clevge of tetrzolium slt present in the regent reflects the mount of ctive cells present in culture. The sornce of the culture mei, which ws correcte for lnk reings in the presence of meium only, ws mesure t A using the Envision Multilel Plte Reer moel 2104 PerkinElmer. Protein etermintion The protein concentrtion of myotue smples ws etermine using the icinchroninic ci (BCA) ssy s escrie erlier y Smith et l. [46]. 2-eoxyglucose uptke Cells seee in 48-well pltes were culture s escrie erlier until the 2 n or 3 r y of ifferentition. They were rinse once with 1xPBS, ph 7.4 (with C 2+,Mg 2+ ) n incute t given time points with experimentl mei (see figure legen). Cells were wshe 3 with HBS solution, n glucose uptke ws crrie out using [1-3 H] 2-DOG s escrie y Klip et l. [47]. Unless stte otherwise, cells were incute with 2-DOG for 30 min. Crrierepenent glucose uptke ws checke using [1-3 H]2- DOG solution contining vrying concentrtion of cyto B [40]. Smples were ssye for [1-3 H] 2-DOGuptke s isintegrtions per min n normlize to the sl level of glucose uptke in control smples. Glycogen synthesis Glycogen nlysis ws crrie out on cells grown in 48- well pltes s escrie y Al-Khlili et l. [36] with some moifictions. On y 2 or 3 of ifferentition, DM3 mei ws replce with experimentl mei. Cells were rinse fterwrs with HBS solution n then incute with fresh HBS solution contining 0.1 mm eftte BSA (n insulin where inicte) for 2 h. Mei for glycogen nlysis (contining [1-14 C]-D-glucose) ws e to the cells uring the lst 1.5 h of incution. Cells were wshe 3 times with ice-col 1xPBS, ph 7.4 (without C 2+,Mg 2+ ) n lyse with 200 μl 30% KOH per well for t lest 30 min. The cell lyste ws trnsferre into eppenorf tues, hete for 10 min t 96 C n 3 mg/ml of crrier glycogen ws e to the cell lyste. A portion of the cell lyste ws use for protein etermintion. Glycogen ws precipitte y overnight incution with 5 volumes of ethnol n 5% soium sulfte t 20 C. The precipitte glycogen ws collecte y centrifugtion t g for 5 min, n reissolve in istille wter. Rioctivity ws mesure in scintilltion mix s isintegrtions per min n the vlues were correcte to the protein content of the cells. Sttisticl nlysis Sttisticl nlyses were performe using the MIXED proceure in SAS version 9.2 (SAS Institute Inc., Cry, NC, USA). The results re presente s lest squre men estimtes (LSmens) n the stnr error of men (SEM). When the min effects were significnt, the lest squre men estimtes were seprte y lest significnt ifference (p < 0.05). All t re expresse s fol chnges normlize to control smples. Ethics pprovl Pigs use for the isoltion of stellite cells were trete ccoring to the Dnish Ministry of Justice Lw, no. 382 (June 10, 1987). Arevitions 2-DOG: 2-eoxyglucose; BCA: Bicinchroninic ci; CLA: Conjugte linoleic ci; cyto B: Cytochlsin B; eftte BSA: Ftty ci-free ovine serum lumin; DM1: First ifferentition mei; DM2: Secon ifferentition mei; DM3: Thir ifferentition mei; DMEM: Dulecco s Moifie Egle s Meium; DMSO: Dimethyl sulfoxie; FBS: Fetl ovine serum; GLUT: Glucose trnsporter; HBS: Hepes-uffere sline; HS: Horse serum; LSmens: Lest squre men estimtes; PA: Phytnic ci; PAM: Plmitic ci; PBS: Phosphte uffere sline; PGM: Prolifertion growth mei; PPAR: Peroxisome prolifertor-ctivte receptor; RXR: Retinoi-X-receptor; SEM: Stnr error of men. Competing interests The uthors eclre tht they hve no competing interests. Authors contriutions BNC esigne n performe experiments, nlyze t n wrote the mnuscript. NO contriute to sttisticl nlysis of t, iscussion, review n eiting of mnuscript. LIH contriute to the conception of the project, iscussion, interprettion, review n eiting of the mnuscript. JHN contriute to the conception of the project. JFY contriute to the experimentl esign, iscussion, review n eiting of mnuscript. All uthors re n pprove the finl mnuscript. Acknowlegements This stuy is prt of the project; Tilore milk for humn helth n ws supporte y the Dnish Council for Strtegic Reserch n The Dnish Cttle Feertion. The uthors thnk Ase Sorensen for proof reing of the mnuscript n Inge-Lise Sørensen n Anne-Grete Dyrvig Peersen for technicl support. Author etils 1 Deprtment of Foo Science, Arhus University, Blichers Allé 20, Tjele 8830, Denmrk. 2 Deprtment of System Biology, Technicl University of Denmrk, 2800 Kgs, Lyngy, Denmrk. 3 Deprtment of Foo Science, Copenhgen University, 1958 Freerikserg C, Copenhgen, Denmrk. Receive: 1 August 2012 Accepte: 31 Jnury 2013 Pulishe: 11 Ferury 2013

11 Che et l. Lipis in Helth n Disese 2013, 12:14 Pge 11 of 12 References 1. Steyn NP, Mnn J, Bennett PH, Temple N, Zimmet P, Tuomilehto J, Linstrom J, Louhernt A: Diet, nutrition n the prevention of type 2 ietes. Pulic Helth Nutr 2004, 7: Wrensjo E, Jnsson JH, Berglun L, Bomn K, Ahren B, Weinehll L, Linhl B, Hllmns G, Vessy B: Estimte intke of milk ft is negtively ssocite with criovsculr risk fctors n oes not increse the risk of first cute myocril infrction. A prospective cse control stuy. Brit J Nutr 2004, 91: Elwoo PC, Pickering JE, Givens DI, Gllcher JE: The consumption of milk n iry foos n the incience of vsculr isese n ietes: n overview of the evience. Lipis 2010, 45: Soemh-Muthu SS, Ding EL, Al-Delimy WK, Hu BF, Engerik FM, Willett WC, Johnn GM: Milk n iry consumption n incience of criovsculr iseses n ll-cuse mortlity: ose response metnlysis of prospective cohort stuies. Am J Clin Nutr 2011, 93: Biong AS, Renor HM, Fimreite RL, Trygg KU, Ringst J, Thelle DS, Peersen JI: Intke of iry ft n iry proucts, n risk of myocril infrction: cse control stuy. Int J Foo Sci Nutr 2008, 59: Wrensjö E, Smemn A, Stegmyr B, Hllmns G, Weinehll L, Essy B, Johsson I: Stroke n plsm mrkers of milk ft intke - prospective neste cse control stuy. J Nutr 2009, 8: Wrensjö E, Jnsson J-H, Ceerholm T, Bomn K, Elisson M, Hllmns G, Johnsson I, Sjögren P: Biomrkers of milk ft n the risk of myocril infrction in men n women: prospective, mtche cse control stuy. Am J Clin Nutr 2010, 92: Wolk A, Furuheim M, Vessy B: Ftty ci composition of ipose tissue n serum lipis re vli iologicl mrkers of iry ft intke in men. J Nutr 2001, 131: Belury MA: Conjugte Dienoic Linolete: polyunsturte ftty ci with unique chemoprotective properties. Nutr Rev 1995, 53: Proi PW: Milk ft in humn nutrition. Aust J Diry Technol 2004, 59: Ellinghus P, Wolfrum C, Assmnn G, Spener F, Seeorf U: Phytnic ci ctivtes the peroxisome prolifertor-ctivte receptor lph (PPAR lph) in sterol crrier protein 2- sterol crrier protein x-eficient mice. J Biol Chem 1999, 274: Kitreewn S, Burk LT, Tomer KB, Prker CE, Detering LJ, Stevens RD, Formn BM, Mis DE, Heymn RA, McMorris T, Weinerger C: Phytol metolites re circulting ietry fctors tht ctivte the nucler receptor RXR. Mol Biol Cell 1996, 7: Lemotte PK, Keiel S, Apfel CM: Phytnic ci is retinoi X receptor lign. Eur J Biochem 1996, 236: Grimli PA: Peroxisome prolifertor-ctivte receptors s sensors of ftty cis n erivtives. Cell Mol Life Sci 2007, 64: Heim M, Johnson J, Boess F, Benik I, Weer P, Hunziker W, Fluhmnn B: Phytnic ci, nturl peroxisome prolifertor-ctivte receptor gonist, regultes glucose metolism in rt primry heptocytes. FASEB J 2002, 16: Hellgren LI: Phytnic ci-n overlooke ioctive ftty ci in iry ft? Ann Ny Ac Sci 2010, 1190: Brown PJ, Mei G, Gier FB, Burston D, Myne PD, Mcclinchy JE, Siey M: Diet n Refsums isese - the etermintion of phytnic ci n Phytol in certin foos n the ppliction of this knowlege to the choice of suitle convenience foos for ptients with Refsums isese. JHumNutrDiet1993, 6: Al-Dirshi OY, Snt T, Rshe MS, Al-Hssnn Z, Shimozw N, Cherwi A, Jco M, Al-Mokh M: Rpi UPLC-MS/MS metho for routine nlysis of plsm pristnic, phytnic, n very long chin ftty ci mrkers of peroxisoml isorers. J Lipi Res 2008, 49: Allen NE, Grce PB, Ginn A, Trvis RC, Rom AW, Appley PN, Key T: Phytnic ci: mesurement of plsm concentrtions y gs liqui chromtogrphy-mss spectrometry nlysis n ssocitions with iet n other plsm ftty cis. Brit J Nutr 2008, 99: Nechi T, Knzki M: Regultion of glucose trnsporters y insulin n extrcellur glucose in C2C12 myotues. Am J Physiol-Enoc M 2006, 291:E817 E Cooksey RC, Heert J, Zhu JH, Woffor P, Grvey WT, McClin DA: Mechnism of hexosmine-inuce insulin resistnce in trnsgenic mice overexpressing glutmine:fructose-6-phosphte miotrnsferse: Decrese glucose trnsporter GLUT4 trnsloction n reversl y tretment with thizoliineione. Enocrinology 1999, 140: Pulw LK, Eckel RH: Overexpression of muscle lipoprotein lipse n insulin sensitivity. Curr Opin Clin Nutr Met Cre 2002, 5: Zomer AWM, vn er Burg B, Jnsen GA, Wners RJA, Poll-The B, vn er Sg PT: Pristnic ci n phytnic ci: nturlly occurring ligns for the nucler receptor peroxisome proliferter-ctivte receptor lph. J Lipi Res 2000, 41: He WM, Brk Y, Hevener A, Olson P, Lio D, Le J, Nelson M, Ong E, Olefsky JM, Evns RM: Aipose-specific peroxisome prolifertor-ctivte receptor gmm knockout cuses insulin resistnce in ft n liver ut not in muscle. P Ntl Ac Sci USA 2003, 100: Verm NK, Singh J, Dey CS: PPAR-gmm expression moultes insulin sensitivity in C2C12 skeletl muscle cells. Brit J Phrmcol 2004, 143: Lio W, Nguyen MTA, Yoshizki T, Fvelyukis S, Ptsouris D, Immur T, Verm IM, Olefsky JM: Suppression of PPAR-gmm ttenutes insulinstimulte glucose uptke y ffecting oth GLUT1 n GLUT4 in 3 T3- L1 ipocytes. Am J Physiol-Enoc M 2007, 293:E219 E Wng YX, Zhng CL, Yu RT, Cho HK, Nelson MC, Byug-Ocmpo CR, Hm J, Kng H, Evns RM: Regultion of muscle fier type n running enurnce y PPAR elt. Plos Biol 2004, 2: McCrty MF: The chlorophyll metolite phytnic ci is nturl rexinoi - Potentil for tretment n prevention of ietes. Me Hypotheses 2001, 56: Crey GB: The swine s moel for stuying exercise-inuce chnges in lipi metolism. Me Sci Sport Exer 1997, 29: Mu M, Oksjerg N, Rehfelt C: Estlishment n conitions for growth n ifferentition of myolst cell line erive from the semimemrnosus muscle of neworn piglets. In Vitro Cell Dev- An 2008, 44: Schönfel P, Wojtczk L: Ftty cis s moultors of the cellulr prouction of rective oxygen species. Free Ricl Bio Me 2008, 45: Schönfel P, Reiser G: Comment concerning the rticle: Phytnic ci impirs mitochonril respirtion through protonophoric ction y Komen et l.: Brnche chin phytnic ci inhiits the ctivity of the mitochonril respirtory chin. Cell Mol Life Sci 2008, 65: Werner LB, Hellgren LI, Rff M, Jensen SK, Petersen RA, Drchmnn T, Tholstrup T: Effect of iry ft on plsm phytnic ci in helthy volunteers - A rnomize controlle stuy. Lipi Helth Dis 2011, 10: Wickn DR, Morit TN: 2-Deoxyglucose; metolic lock for glucose. Proc Soc Exp Biol Me 1955, 89: Mueckler M: Fmily of glucose-trnsporter genes. Implictions for glucose homeostsis n ietes. Dietes 1990, 39: Al-Khlili L, Chilin AV, Knnisto K, Zhng BB, Permert J, Holmn GD, Ehrenorg E, Ding VDH, Zierth JR, Krook A: Insulin ction in culture humn skeletl muscle cells uring ifferentition: ssessment of cell surfce GLUT4 n GLUT1 content. Cell Mol Life Sci 2003, 60: Estensen RD, Plgemnn PG: Cytochlsin B: inhiition of glucose n glucosmine trnsport. P Ntl Ac Sci USA 1972, 69: Chn YJ, Amrit LR: Cytochlsin B ining sites n glucose trnsport crrier in humn erythrocytes ghosts. J Biol Chem 1977, 252: Schürmnn A: Insight into the o hexose trnsporters GLUT3, GLUT5, n GLUT7. Am J Physiol-Enoc M 2008, 295:E225 E Sri V, Lm L, Burett E, Leiter LA, Klip A: Glucose-trnsport in humn skeletl-muscle cells in culture - stimultion y insulin n metformin. J Clin Invest 1992, 90: Archulet TL, Lemieux AM, Sengsirisuwn V, Techey MK, Linorg KA, Kim JS, Henriksen EJ: Oxint stress-inuce loss of IRS-1 n IRS-2 proteins in rt skeletl muscle: Role of p38 MAPK. Free Ricl Bio Me 2009, 47: Kim JK, Zismn A, Fillmore JJ, Peroni OD, Kotni K, Perret P, Zong H, Dong J, Khn CR, Khn BB, Shulmn GI: Glucose toxicity n the evelopment of ietes in mice with muscle-specific inctivtion of glut4. J Clin Invest 2001, 108: Shepher PR, Khn BB: Mechnisms of isese - glucose trnsporters n insulin ction - implictions for insulin resistnce n ietes mellitus. New Engl J Me 1999, 341: Theil PK, Sorensen IL, Nissen PM, Oksjerg N: Temporl expression of growth fctor genes of primry porcine stellite cells uring myogenesis. J Anim Sci 2006, 77:

12 Che et l. Lipis in Helth n Disese 2013, 12:14 Pge 12 of Okur T, Igse M, Kitmi Y, Fukuok T, Mguchi M, Kohr K, Hiw K: Pltelet-erive growth fctor inuces poptosis in vsculr smooth muscle cells: roles of the Bcl-2 fmily. BBA-Mol Cell Res 1998, 1403: Smith PK, Krohn RI, Hermnson GT, Mlli AK, Grtner FH, Provenzno MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC: Mesurement of protein using incinchoninic ci. Anl Biochem 1985, 150: Klip A, Rmll T: Protein-kinse-C is Not require for insulin stimultion of hexose uptke in muscle-cells in culture. Biochem J 1987, 242: oi: / x Cite this rticle s: Che et l.: Phytnic ci stimultes glucose uptke in moel of skeletl muscles, the primry porcine myotues. Lipis in Helth n Disese :14. Sumit your next mnuscript to BioMe Centrl n tke full vntge of: Convenient online sumission Thorough peer review No spce constrints or color figure chrges Immeite puliction on cceptnce Inclusion in PuMe, CAS, Scopus n Google Scholr Reserch which is freely ville for reistriution Sumit your mnuscript t