Insulin signaling: A potential signaling pathway for the stimulatory effect of kaempferitrin on glucose uptake in skeletal muscle

European Journal of Pharmacology ] (]]]]) ]]]–]]]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66

Contents lists available at SciVerse ScienceDirect

European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar

Full length article

Insulin signaling: A potential signaling pathway for the stimulatory effect of kaempferitrin on glucose uptake in skeletal muscle

Q1

Luisa Helena Cazarolli b, Danielle Fontana Pereira a, Virginia Demarchi Kappel a, Poliane Folador a, Maria dos Santos Reis Bonorino Figueiredo a, Moacir Geraldo Pizzolatti c, Fa´tima Regina Mena Barreto Silva a,n a

´rio, Bairro Trindade, Cx. Postal 5069, ´gicas, Universidade Federal de Santa Catarina, Campus Universita Departamento de Bioquı´mica, Centro de Ciˆencias Biolo ´polis, SC, Brazil CEP 88040-970 Floriano b ´rio Laranjeiras do Sul, Bairro Vila Alberti, CEP 85303-775, Laranjeiras do Sul, PR, Brazil Universidade Federal da Fronteira Sul, Campus Universita c ´ticas, UFSC, Campus Universita ´rio, Bairro Trindade, Cx. Postal 5069, CEP 88040-970 Floriano ´polis, SC, Brazil Departamento de Quı´mica, Centro de Ciˆencias Fı´sicas e Matema

a r t i c l e i n f o

abstract

Article history: Received 4 October 2012 Received in revised form 8 February 2013 Accepted 14 February 2013

The aim of the study was to investigate the in vitro effect and the mechanism of action of kaempferitrin on glucose uptake in an insulin target (soleus muscle). A stimulatory effect of kaempferitrin on glucose uptake was observed when rat soleus muscle was incubated with 10, 100 and 1000 ZM of this flavonoid glycoside. The presence of specific insulin signaling inhibitors, such as wortmannin, an inhibitor of phosphoinositide 3-kinase (PI3K), RO318220, an inhibitor of protein kinase C (PKC), PD98059, an inhibitor of mitogen-activated protein kinase (MEK), HNMPA(AM)3, an insulin receptor tyrosine kinase activity inhibitor, colchicine, a microtubule-depolymerizing agent, SB239063, an inhibitor of P38 MAPK and cycloheximide, an inhibitor of protein synthesis showed that kaempferitrin triggers different metabolic and nuclear pathways in skeletal muscle. Besides the influence on glycogen storage, the metabolic action involves the insulin receptor, PI3K, atypical PKC activity and the translocation of GLUT4. Additionally, the nuclear pathways (via MAPK and MEK) provide evidence of the stimulation of the expression of glucose transporters or other signaling proteins, reinforcing proposals that skeletal muscle represents a primary site at which kaempferitrin exerts its effect promoting glucose homeostasis. Also, these similarities with the signaling pathways of insulin constitute strong evidence for the insulin-mimetic role of kaempferitrin in glucose homeostasis. & 2013 Published by Elsevier B.V.

Keywords: Kaempferitrin Mechanism of action Glucose uptake Insulin Diabetes Skeletal muscle

1. Introduction Insulin is the most important physiological stimulus of glucose uptake in skeletal muscle. It induces a redistribution of GLUT4 glucose transporter proteins from the cell interior to the surface plasma membrane, which ultimately leads to a greater glucose uptake rate in the muscle cells (Klip, 2009). The increase in GLUT4 exocytose in response to insulin is triggered through signaling via the insulin receptor that induces downstream activation of signaling pathways such as PI3K/AKT/PKB, PKCs, CAP/CBL/TC10 and MAPKs (Chang et al., 2004; Kanzaki, 2006; Klip, 2009). An absolute or relative lack of insulin, as in the case of diabetes, leads to severe dysfunction and deregulation of insulin signaling in target tissues such as the muscle, adipose tissue and liver. Diabetes mellitus is a metabolic disorder characterized by chronic hyperglycemia resulting from defects in insulin secretion, insulin action, or both (American Diabetes

n

Q1

Corresponding author. Tel.: þ55 48 3721 69 12; fax: þ 55 48 3721 96 72. E-mail address: [email protected] (R.M.B. Silva).

Association, 2008). The molecular defects accounting for impaired glucose utilization are not fully understood but may involve defective GLUT4 translocation, glucose uptake and aberrant insulin signal transduction. In this context, several naturally occurring polyphenols have been shown to affect glucose uptake and insulin-receptor function, both of which play an essential role in diabetes (Cazarolli et al., 2008, 2009a; Zanatta et al., 2008; Jung et al., 2008). Kaempferitrin, a 3,7-diglycosylflavone, has been described as an antimicrobial, antiinflammatory and antioxidant agent (Abdel-Ghani et al., 2001; De Sousa et al., 2004; Fang et al., 2005). Also, we have previously demonstrated that this flavonoid can reduce serum glucose levels in diabetic rats and increase glucose uptake in soleus muscle (De Sousa et al., 2004; Jorge et al., 2004; Cazarolli et al., 2006). Additionally, kaempferitrin has been shown to stimulate GLUT4 translocation and synthesis in adipocytes and this effect involves, at least in part, the classical insulin signaling pathway and adiponectin secretion (Tzeng et al., 2009). Although the potential insulinomimetic effect of kaempferitrin has been previously reported (De Sousa et al., 2004; Jorge et al., 2004; Cazarolli et al., 2006) the mechanisms involved in the stimulatory action of this flavonoid on skeletal muscle and on

0014-2999/$ - see front matter & 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.ejphar.2013.02.029

Please cite this article as: Cazarolli, L.H., et al., Insulin signaling: A potential signaling pathway for the stimulatory effect of kaempferitrin on glucose uptake in skeletal muscle. Eur J Pharmacol (2013), http://dx.doi.org/10.1016/j.ejphar.2013.02.029i

67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91

2

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66

L.H. Cazarolli et al. / European Journal of Pharmacology ] (]]]]) ]]]–]]]

glucose uptake should be elucidated. In this study, the mechanism of action of kaempferitrin associated with the stimulatory effect on [14C]-D-glucose uptake in soleus muscle was investigated.

2. Materials and methods 2.1. Materials Regular human insulin (Biohulin) was obtained from Biobra´s, Bioquı´mica do Brasil S/A (A´guas Claras, MG, Brazil). [U-14C]-2Deoxy-D-glucose ([14C]DG), specific activity 10.6 GBq/mmol, D-[14C (U)]-glucose ([14C]-glucose), specific activity 9.25 GBq/mmol, thymidine [methyl-14C], specific activity 1.7464 GBq/mmol and biodegradable scintillation liquid were obtained from Perkin–Elmer Life and Analytical Sciences (Boston, MA, USA). Wortmannin [inhibitor of phosphoinositide 3-kinase (PI3K)], RO318220 (2-{1-[3-(amidinothio)propyl]-1H-indol-3-yl}-3-(1-methylindol-3-yl)maleimide methanesulfonate) [inhibitor of protein kinase C (PKC)], PD98059 (2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one) [inhibitor of mitogen-activated protein kinase (MEK)], cycloheximide (3-[2(3,5-dimethyl-2-oxocyclohexyl)-2-hydroxyethyl]glutarimide) [inhibitor of protein synthesis], SB239063 (trans-1-(4-hydroxycyclohexyl)-4-(4-fluorophenyl)-5-(2-methoxypyridimidin-4-yl)imidazole) [inhibitor of P38MAPK] and colchicine [microtubuledepolymerizing agent] were purchased from Sigma Chemical Companys (St. Louis, MO, USA). HNMPA(AM)3 (hydroxy-2-naphthalenylmethylphosphonic acid tris-acetoxy-methyl ester) [inhibitor of insulin receptor tyrosine kinase activity] was purchased from Enzo Life Sciencess (NY, USA). Salts and solvents were purchased from Merck AG (Darmstadt, Germany). 2.2. Plant material The leaves of Bauhinia forficata Link were collected in the summer of 1999 in Orleans, Brazil, and identified by Prof. Daniel de Barcelos Falkenberg. A voucher specimen of the plant (FLOR31271) has been deposited in the herbarium of the Botany department at Universidade Federal de Santa Catarina, Floriano´polis, Brazil. The process of kaempferitrin extraction and isolation was carried out as described in De Sousa et al. (2004). The flavonoid used in this study was dissolved in 1% EtOH/H2O solution and stored at  20 1C. 2.3. Experimental animals Male Wistar rats weighing 180–200 g from the Central Animal House-UFSC were used. The rats were housed in plastic cages in an air-conditioned animal room and fed on pellets with free access to tap water. Room temperature was controlled at 21 1C with a 12 h light:12 h dark cycle. Animals described as fasted had been deprived of food for 16 h but allowed free access to water. All the animals were monitored carefully and maintained in accordance with the ethical recommendations of the Brazilian Veterinary Medicine Council and the Brazilian College of Animal Experimentation. (Protocol CEUA/PP007). Rats fasted for 16 h received 50 mg/kg body weight of alloxan (Sigma, St. Louis, MO, USA) by a single intravenous injection. The diabetic state was assessed by measuring body weight and blood glucose levels 3 days later (Jorge et al., 2004). 2.4. Studies on [14C]-glucose uptake in rat soleus muscle For the [U-14C]-2-deoxy-D-glucose uptake experiments, soleus muscles from normal rats were used. Slices of soleus muscle were distributed (alternately left and right) between basal and treated

groups. The muscles were dissected, weighed, and preincubated and incubated at 37 1C in Krebs Ringer-bicarbonate (KRb) buffer with a composition of 122 mM NaCl, 3 mM KCl, 1.2 mM MgSO4, 1.3 mM CaCl2, 0.4 mM KH2PO4, and 25 mM NaHCO3 and bubbled with O2/CO2 (95:5 v/v%) until pH 7.4. Kaempferitrin (compound 1) (0.001, 0.01, 0.1, 1, 10, 100 and 1000 ZM) and insulin (10 ZM) were added to the pre-incubation (30 min) and incubation media (60 min) in the presence or absence of 100 ZM wortmannin, 40 mM RO318220, 50 mM PD98059, 0.35 mM cycloheximide, 100 mM HNMPA(AM)3, 10 mM SB239063 or 1 mM colchicine. [14C]DG (0.1 mCi/ml) was added to each sample during the incubation period. After incubation, the muscle samples were homogenized in 0.5 N NaOH and boiled for 10 min; 25 ml aliquots of tissue and external medium were placed in scintillation liquid on an LKB RackBeta liquid scintillation spectrometer (model 1215; EG and G-Wallac, Turku, Finland), for the radioactivity measurements. The results were expressed as the tissue/medium (T/M) ratio: cpm/ml tissue fluid per cpm/ml incubation medium (Jorge et al., 2004).

2.5. Studies on thymidine incorporation into rat soleus muscle For the [methyl-14C]-thymidine uptake experiments, soleus muscles from normal rats were used. Slices of soleus muscle were distributed (alternately left and right) between basal and treated groups. The muscles were dissected, weighed, and preincubated and incubated at 37 1C in KRb buffer with a composition of 122 mM NaCl, 3 mM KCl, 1.2 mM MgSO4, 1.3 mM CaCl2, 0.4 mM KH2PO4, and 25 mM NaHCO3 and bubbled with O2/CO2 (95:5 v/v%) until pH 7.4. Kaempferitrin (compound 1) (10 ZM) was added to the pre-incubation (30 min) and incubation (60 min) media. [14C]thymidine (0.5 mCi/ml) was added to each sample during the incubation period. After incubation, muscle samples were washed in cold KRb and dried on filter paper. Cold trichloroacetic acid (TCA) was then added to each tube to give a final concentration of 10% followed by cooling in an ice bath for 2 h. The muscle samples were homogenized and centrifuged at 1800  g for 15 min, the supernatant was discarded and pellets resolubilized in 0.5 N NaOH. The total protein in the tubes was measured according to the method of Lowry et al.; 25 ml aliquots of tissue and external medium were placed in scintillation liquid on an LKB RackBeta liquid scintillation spectrometer (model 1215; EG and G-Wallac, Turku, Finland), for the radioactivity measurements. The results for the protein synthesis were expressed as the cpm/mg protein ratio.

2.6. Studies on glycogen content in rat soleus muscle Soleus muscles were harvested from the untreated diabetic rats (control), treated with kaempferitrin (100 mg/kg) and used for the assay of glycogen content immediately after 3 h of treatment. Glycogen was isolated from this tissue as described by Krisman (1962), with minor modifications (Folador et al., 2010). The tissue was weighed, homogenized in 33% KOH and boiled at 100 1C for 20 min, with occasional stirring. After cooling, 96% ethanol was added to the samples which were then heated to boiling followed by cooling in an ice bath to aid the glycogen precipitation. The homogenate was centrifuged at 1300  g for 15 min, the supernatant was discarded and the pellet was neutralized with saturated NH4Cl before being heated to 100 1C for 5 min, washed and resolubilized in water. Glycogen content was determined by treatment with iodine reagent and the absorbance was measured at 460 nm. The results were expressed as mg of glycogen/g of tissue.

Please cite this article as: Cazarolli, L.H., et al., Insulin signaling: A potential signaling pathway for the stimulatory effect of kaempferitrin on glucose uptake in skeletal muscle. Eur J Pharmacol (2013), http://dx.doi.org/10.1016/j.ejphar.2013.02.029i

67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132

L.H. Cazarolli et al. / European Journal of Pharmacology ] (]]]]) ]]]–]]]

2.7. Studies on glycogen synthesis in rat soleus muscle The assays of [14C]-glucose incorporation into glycogen were conducted as described in Cazarolli et al. (2009b). Slices of soleus muscle from normal rats were distributed (alternately left and right) between control and treated groups. Muscles were dissected, weighed and then preincubated and incubated at 37 1C in KRb buffer comprising 122 mM NaCl, 3 mM KCl, 1.2 mM MgSO4, 1.3 mM CaCl2, 0.4 mM KH2PO4, 25 mM NaHCO3 plus 1% BSA and 5 mM D-glucose and bubbled with O2/CO2 (95:5 v/v%) until pH 7.4 was reached. Kaempferitrin (0.1; 10 and 100 ZM) was added to the pre-incubation (30 min) and incubation (60 min) media. [14C]-glucose (0.15 mCi/ml) was added to each sample during the incubation period. After incubation, the muscle samples were removed, washed in cold KRb and dried on filter paper. The muscle samples were homogenized in 0.5 N KOH, and boiled at 100 1C for 20 min, with occasional stirring. After cooling, 95% ethanol was added to the samples, which were heated to boiling followed by cooling in an ice bath for 20 min to allow glycogen precipitation. The homogenates were centrifuged at 664  g for 15 min, the supernatant was discarded and the pellets resolubilized in water. Aliquots (30 ml) of the samples were placed in liquid scintillation vials in an LKB rack, on a beta liquid scintillation spectrometer (model 1215; EG and G-Wallac, Turku, Finland), for the radioactivity measurements. The results were expressed as pmol glycosyl units incorporated in glycogen/mg tissue/h.

2.8. Data and statistical analysis Data were expressed as mean 7S.E.M. The statistical analysis was carried out using one-way analysis of variance (ANOVA), followed by the Bonferroni post test or non-paired Student’s t test to identify significantly different groups. Differences were considered to be significant at Pr0.05.

Fig. 1. (A) Concentration–response curve of kaempferitrin (compound 1) and (B) effect of 100 mM HNMPA(AM)3, inhibitor of insulin receptor, on the stimulatory action of 10 ZM insulin and 10 ZM kaempferitrin on [14C]-glucose uptake in rat soleus muscle. Pre-incubation time ¼30 min; incubation time¼ 60 min. Values are expressed as mean 7 SEM; n¼ 6 in duplicate for each group. Significant at ***Pr 0.001, **Pr 0.01 and *P r0.05 in relation to basal group; ##Pr 0.01 in relation to insulin group; @@P r0.01 in relation to kaempferitrin group.

3. Results Fig. 1A shows the in vitro effect of kaempferitrin (0.001, 0.01, 0.1, 1, 10, 100 and 1000 ZM) and insulin (10 ZM) on glucose uptake in the rat soleus muscle following 60 min of incubation. The stimulatory effect of kaempferitrin was significant at 10, 100 and 1000 ZM and represented 22, 10 and 11% of glucose uptake compared to the basal value at 60 min, respectively. Based on that, we investigated whether the action of kaempferitrin on glucose uptake involves the insulin signaling pathways. To do this, we performed the glucose uptake assays in the presence of specific inhibitors of insulin signaling. As observed in Fig. 1B, the stimulatory effects of both kaempferitrin and insulin were completely blocked in the presence of 100 mM of HNMPA(AM)3 (Saperstein et al., 1989; Diaz et al., 2007; Gu et al., 2010), an inhibitor of insulin receptor tyrosine kinase activity. The effect of insulin on glucose uptake is mediated through the activation/action of PI3K and 3-phosphoinositide-dependent protein kinases (PDKs) and their downstream effectors such as atypical PKC isoforms (z, l/i) and PKB. The pretreatment of the muscle with wortmannin (PI3K inhibitor) and RO318220 (atypical PKCs inhibitor) completely blocked the kaempferitrin-induced glucose uptake (Fig. 2). Additionally, colchicine, a microtubule-depolymerizing agent, totally blocked the stimulatory effect of kaempferitrin on glucose uptake. In order to more precisely characterize the metabolic effects of kaempferitrin we used a second generation p38 MAPK inhibitor, SB239063 (Underwood et al., 2000; Barone et al., 2001). As shown in Fig. 2, the stimulatory effect of the flavonoid was inhibited in the presence of SB239063. This result indicates that

3 14 C-DG uptake in soleus muscle (T/M)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66

3

***

*** #

#

#

###

2

1

0 Insulin Kaempferitrin Wortmannin RO318220 Colchicine SB239063

-

+ -

+ -

+ -

+ + -

+ -

+ + -

+ -

+ + -

+

+ +

Fig. 2. Effect of enzyme inhibitors, 1 mM colchicine, 10 mM SB239063, 100 ZM Wortmannin, 40 mM RO318220 on the stimulatory action of 10 ZM kaempferitrin (compound 1) on [14C]-glucose uptake in rat soleus muscle. Basal group ¼ no treatment. Signal (þ ) and (  ) indicates presence and absence, respectively, of each substance in the incubation medium. Pre-incubation time ¼ 30 min; incubation time ¼60 min. Values are expressed as mean 7 S.E.M.; n¼ 6 in duplicate for each group. Significant to ***P r 0.001 in relation to basal group. Significant to ### Pr 0.001 and #P r0.05 in relation to kaempferitrin (1) group.

Please cite this article as: Cazarolli, L.H., et al., Insulin signaling: A potential signaling pathway for the stimulatory effect of kaempferitrin on glucose uptake in skeletal muscle. Eur J Pharmacol (2013), http://dx.doi.org/10.1016/j.ejphar.2013.02.029i

67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132

4

besides GLUT4 translocation to the plasma membrane kaempferitrin can also mediate GLUT4 activation. Additionally, insulin plays an important role in the overall regulation of protein synthesis. To determine whether the kaempferitrin effect on glucose uptake involves protein synthesis and/or its action on the cell nucleus we performed an assay on thymidine incorporation in rat soleus muscle (Fig. 3A). In the presence of the flavonoid, thymidine incorporation was increased when compared to the control group. Also, to address the possibility that the MEK inhibitor influences the glucose uptake activity of kaempferitrin, we examined the effect of PD-98059 on kaempferitrin-stimulated glucose uptake (Fig. 3B). This inhibitor completely blocked the increase in glucose uptake compared with the control group. Kaempferitrin may act through a MAPK pathway stimulating the expression of glucose transporters or proteins from the insulin phosphorylation cascades, since protein synthesis was completely inhibited in the presence of cycloheximide (Fig. 3B). Another important pathway of the action of insulin in metabolism regulation is glycogen synthesis. As shown in Fig. 4A, kaempferitrin increased glycogen content in diabetic rats compared with control groups. The known stimulatory effect of insulin on glycogen storage was observed after 3 h (437%) of insulin treatment in diabetic rats compared with the untreated diabetic rats. In percentage terms, the effect of kaempferitrin on the glycogen content of diabetic rats represents an increase of 228% compared with the untreated diabetic group. Additionally, this flavonoid stimulated in vitro glycogen synthesis in soleus muscle compared with the basal glycogen content (Fig. 4B).

4. Discussion We have previously demonstrated the effect of kaempferitrin as an efficient hypoglycemic agent (De Sousa et al., 2004; Jorge Basal

14 C-Thymidine DNA incorporation (cpm/μg protein)

3

Kaempferitrim 10 ηM * 2

1

0

C-DG uptake in soleus muscle (T/M)

3 **

** ###

###

2

1

14

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66

L.H. Cazarolli et al. / European Journal of Pharmacology ] (]]]]) ]]]–]]]

0 Insulin Kaempferitrin Cycloheximide PD98059

-

+ -

+ -

+ -

+ + -

+

+ +

Fig. 3. (A) Effect of kaempferitrin (compound 1) on thymidine incorporation and (B) effect of enzyme inhibitors 50 mM PD98059 and 0.35 mM cycloheximide on the stimulatory action of 10 ZM kaempferitrin (compound 1) on [14C]-glucose uptake in rat soleus muscle. Basal group ¼no treatment. Signal (þ ) and (  ) indicates presence and absence, respectively, of each substance in the incubation medium. Pre-incubation time ¼30 min; incubation time¼ 60 min. Values are expressed as mean 7S.E.M.; n¼6 in duplicate for each group. Significant to *Pr 0.05 and **P r0.01 in relation to basal group. Significant to ###Pr 0.001 in relation to kaempferitrin (compound 1) group.

Fig. 4. (A) Effect of kaempferitrin (1) (100 mg/kg) and insulin (0.5 IU) on muscle glycogen content in diabetic rats and (B) dose–response curve of kaempferitrin in glycogen synthesis in the rat soleus muscle. Pre-incubation time ¼ 30 min; incubation time ¼60 min. Values are expressed as mean 7 S.E.M.; n¼6 in duplicate for each group. Significant at **P r0.01 in relation to diabetic control group and ***P r0.001 in relation to basal control group.

et al., 2004). However, its mechanism of action in relation to glucose uptake has not been determined. Thus, this study was designed to investigate the mechanism of insulin-mimetic activity of kaempferitrin on glucose uptake in soleus muscle. Our study demonstrated that 10 ZM kaempferitrin acutely stimulated the glucose uptake in soleus muscle. In percentage terms, kaempferitrin presented an effect similar to that of 10 ZM insulin on glucose uptake. This result is in line with those published concerning the hypoglycemic effect of kaempferol 3-neohesperidoside and kaempferitrin in diabetic rats and the stimulatory effect of these flavonoids on glucose uptake (Jorge et al., 2004; Cazarolli et al., 2006; Zanatta et al., 2008; Yamasaki et al., 2011). Thus, we investigated whether the action of kaempferitrin on glucose uptake involves the insulin signaling pathways and it was observed that kaempferitrin’s stimulatory effect was completely blocked by HNMPA(AM)3, an inhibitor of insulin receptor tyrosine kinase activity. In skeletal muscle, the signal transduction is mediated by a series of phosphorylation cascades linking the initial activation of the insulin receptor (IR) tyrosine kinase activity to downstream substrates (Taha and Klip, 1999; Chang et al., 2004; Krook et al., 2004; Kanzaki, 2006). It has been demonstrated that the presence of HNMPA(AM)3 can totally block the stimulatory effect of insulin on glucose uptake and on glucose oxidation in CHO cells (Yamasaki et al., 2011). To date only a few naturally derived compounds have been identified as acting on and/or enhancing insulin receptor

Please cite this article as: Cazarolli, L.H., et al., Insulin signaling: A potential signaling pathway for the stimulatory effect of kaempferitrin on glucose uptake in skeletal muscle. Eur J Pharmacol (2013), http://dx.doi.org/10.1016/j.ejphar.2013.02.029i

67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132

L.H. Cazarolli et al. / European Journal of Pharmacology ] (]]]]) ]]]–]]]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66

phosphorylation and these include metformin, fenugreek seed extract, demethylasterriquinone B-l (DMAQ-B1) and TLK1699825 (Salituro et al., 2001; Vijayakumar et al., 2005; Bailey, 2007). Furthermore, kaempferitrin was recently demonstrated to upregulate and increase the phosphorylation of the insulin receptor beta subunit in 3T3-L1 adipocytes (Tzeng et al., 2009). Considered together, these results reinforce the potential effect of kaempferitrin as an insulin mimetic agent. This is significant since the structure of flavonoids has been reported to be associated with insulin receptor activities (Cazarolli et al., 2008). The effect of insulin on glucose uptake is mediated through the activation/action of PI3K and 3-phosphoinositide-dependent protein kinases (PDKs) and their downstream effectors such as atypical PKC isoforms (z, l/i) and PKB. The pretreatment of the muscle with wortmannin (PI3K inhibitor) and RO318220 (atypical PKCs inhibitor) completely blocked the kaempferitrin-induced glucose uptake (Fig. 2). The activation of PKB by insulin via PI3K has been described as essential for glucose transporter translocation to the plasma membrane (Hajduch et al., 2001; Kanzaki, 2006). Besides the involvement of the PI3K pathway, the activity of atypical PKC isoforms (z, l, i) also plays a role in the stimulatory action of kaempferitrin on glucose uptake. It has been demonstrated that the activation of aPKCs is dependent on the PDK1 activity. Activated aPKCs were shown to be physically associated with GLUT4 compartments. Additionally, aPKCs directly phosphorylate GLUT4-compartment-associated proteins in serine/threonine residues, such as VAMP2, resulting in increased GLUT4 translocation and glucose uptake (Braiman et al., 2001; Watson et al., 2004; Kanzaki et al., 2004; Liu et al., 2006). Recently, kaempferitrin was demonstrated to increase AKT phosphorylation and GLUT4 translocation in 3T3-adipocytes, which were inhibited by wortmannin (Tzeng et al., 2009). These observations suggest that muscle cells are targets for kaempferitrin action. Also, these results reinforce proposals that kaempferitrin stimulates glucose uptake through an insulin signaling pathway involving PI3K-PKB and aPKC activation, and probably interferes with other proteins and structures related to GLUT4 translocation to the plasma membrane. In the case of GLUT4, the actin and microtubule cytoskeleton networks have been implicated in the subcellular movements of Q2 the GLUT4-containing vesicles (Lane and Allan, 1998; Watson et al., 2004; Kanzaki, 2006; Chen et al., 2008). It has been suggested that insulin stimulation can induce release of vesicles allowing their movement to the plasma membrane by simple diffusion, or insulin may trigger the movement of vesicles along cytoskeletal tracks (Watson et al., 2004; Kanzaki, 2006). In this way, we studied the effect of colchicine, a microtubuledepolymerizing agent that totally blocked the stimulatory effect of kaempferitrin on glucose uptake. It has been demonstrated that the disruption of microtubules caused by colchicine and other microtubule-depolymerizing agents inhibits insulin-induced GLUT4 translocation and glucose uptake in adipocytes (Fletcher et al., 2000; Olson et al., 2001; Watson et al., 2004; Huang et al., 2005). The inhibition of the stimulatory effect of kaempferitrin on glucose uptake by colchicine suggests that for the complete and efficient effect of this flavonoid on glucose uptake to occur cytoskeleton integrity is required. Muscle and fat tissues are the main sites responsible for glucose uptake in response to insulin in vivo and GLUT4 is the predominant glucose transporter isoform expressed in these tissues. However, a discrepancy between the magnitude of GLUT4 translocation and glucose uptake has often been observed. These findings indicate that there is an increase in the intrinsic activity of GLUT4 beyond induced-translocation (Furtado et al., 2003). Moreover, recent evidence has implicated p38 MAPK in GLUT4 activation by insulin, probably influencing its phosphorylation

5

state at the plasma membrane (Furtado et al., 2003). In order to more precisely characterize the metabolic effects of kaempferitrin we used a second generation p38 MAPK inhibitor, SB239063 (Underwood et al., 2000; Barone et al., 2001) that inhibited kaempferitrin’s stimulatory effect on glucose uptake. This result indicates that besides GLUT4 translocation to the plasma membrane kaempferitrin can also mediate GLUT4 activation. It has previously been shown that the SB202190 and SB203580 (structurally similar inhibitors of p38 MAPK) attenuate glucose uptake without affecting the ability of GLUT4 translocation to the cell surface in response to insulin (Sweeney et al., 1999; Somwar et al., 2001). Based on the results discussed above in relation to kaempferitrin, we propose that this flavonoid increases glucose uptake in the skeletal muscle via multiple mechanisms. This involves the PI3K and atypical PKCs pathways and also may involve PI3Kindependent but complementary pathways, such as p38 MAPK, reinforcing suggestions that skeletal muscle represents a primary site in which kaempferitrin exerts its effects. Insulin plays an important role in the overall regulation of protein synthesis. Some of the effects of the hormone involve changes in the abundance of mRNAs, but insulin also has important effects on the translation process itself (Taha and Klip, 1999). In the presence of kaempferitrin, thymidine incorporation was increased when compared to the control group. Also, insulin triggers signaling pathways that participate broadly in cellular growth and differentiation, such as the MAPK pathways (Taha and Klip, 1999). Taking this into account, the effect of PD-98059 on kaempferitrin-stimulated glucose uptake was studied and it was demonstrated that this inhibitor completely blocked the increase in glucose uptake compared with the control group. The involvement of MAPK in glucose transport has also been described for apigenin-6-C-(200 -O-a-L-rhamnopyranosyl)-bL-fucopyranoside and nitroprusside, since PD98059 suppresses this effect (Jensen et al., 2003; Cazarolli et al., 2009a). Kaempferitrin may act through a MAPK pathway stimulating the expression of glucose transporters or proteins from the insulin phosphorylation cascades, since protein synthesis was completely inhibited in the presence of cycloheximide. Additionally, extracts from Aegles marmelos, Syzygium cumini and Canna indica, plants rich in flavonoids, stimulated glucose uptake and these effects were totally inhibited in the presence of cycloheximide, suggesting that active protein synthesis is important to maintaining appropriate glucose transport (Anandharajan et al., 2006; Purintrapiban et al., 2006). Thus, kaempferitrin-stimulated glucose transport is mediated through active protein synthesis and, coupled with the involvement of PI3K, PKC and p38 MAPK pathways, reinforces the insulinomimetic effect of this flavonoid in an insulin-sensitive tissue, soleus muscle. Another important pathway of the action of insulin in metabolism regulation is glycogen synthesis. Insulin regulates glycogen synthase activation by controlling the uptake and transport by GLUT4 of glucose and by regulating the phosphorylation and activation states of enzymes involved in the synthesis and degradation of glycogen (Srivastava and Pandey, 1998). Kaempferitrin increased glycogen content in diabetic rats compared with control groups and also significantly stimulated in vitro glycogen synthesis in soleus muscle. Besides kaempferitrin effect on in vivo glycogen content, this flavonoid surprisingly presented a stimulatory effect on in vitro glycogen synthesis in a more powerful dose than that reported to insulin in a similar experimental approach (Cazarolli et al., 2009b). Also, kaempferitrin presented a quite similar effectiveness considering the range of concentrations (0.1 ZM–10 ZM) in both, glucose uptake and glycogen synthesis. Previous studies have shown that flavonoids, such as catechin, myricetin, kaempferol 3-neohesperidoside and apigenin-6-C-b-L-fucopyranoside, act

Please cite this article as: Cazarolli, L.H., et al., Insulin signaling: A potential signaling pathway for the stimulatory effect of kaempferitrin on glucose uptake in skeletal muscle. Eur J Pharmacol (2013), http://dx.doi.org/10.1016/j.ejphar.2013.02.029i

67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132

6

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66

L.H. Cazarolli et al. / European Journal of Pharmacology ] (]]]]) ]]]–]]]

in the insulin signaling pathways that regulate glucose uptake and glycogen synthesis. These flavonoids can stimulate glycogen synthesis and glucose uptake in insulin target tissues, such as soleus muscle and liver tissues (Ong and Khoo, 1996, 2000; Valsa et al., 1997; Cazarolli et al., 2009b, 2009c).

5. Conclusions Kaempferitrin stimulates glucose uptake in soleus muscle via classical insulin signaling pathways. These results indicate the role of the PI3K and MAPK pathways in the stimulatory effect of kaempferitrin on glucose uptake involving, at least, the translocation and synthesis of GLUT4. Moreover, in the insulin target tissue investigated (soleus muscle) this flavonoid stimulates glycogen synthesis. Kaempferitrin triggers multiple stimulatory pathways in skeletal muscle in the regulation of glucose homeostasis.

Acknowledgment This work was supported by grants from Conselho Nacional de Desenvolvimento e Tecnolo´gico (CNPq), Coordenac- a~ o de Pessoal de Nı´vel Superior (CAPES-PGFAR; CAPES-PG-Biochemistry), and Fundac- a~ o de Amparo a Pesquisa do Estado de Santa Catarina (FAPESC). The authors express their appreciation to Dr. Siobhan Wiese for assistance with the English correction of the manuscript. References Abdel-Ghani, N.T., Shoukry, A.F., el Nashar, R.M., 2001. Flow injection potentiometric determination of pipazethate hydrochloride. Analyst 126, 79–85. American Diabetes Association, 2008. Diagnosis and classification of diabetes mellitus. Diab. Care, 31, S55 – S60. Anandharajan, R., Jaiganesh, S., Shankernarayanan, N.P., Viswakarma, R.A., Balakrishnan, A., 2006. In vitro glucose uptake activity of Aegles marmelos and Syzygium cumini by activation of GLUT-4, PI3 kinase and PPARg in L6 myotubes. Phytomedicine 13, 434–441. Bailey, C.J., 2007. Treating insulin resistance: future prospects. Diab. Vasc. Dis. Res. 4, 20–31. Barone, F.C., Irving, E.A., Ray, A.M., Lee, J.C., Kassis, S., Kumar, S., Badger, A.M., White, R.F., Mcvey, M.J., Legos, J.J., Erhardt, J.A., Nelson, A.H., Ohlstein, E.H., Hunter, A.J., Ward, K., Smith, B.R., Adams, J.L., Parsons, A.A., 2001. SB 239063, a second-generation p38 mitogen-activated protein kinase inhibitor, reduces brain injury and neurological deficits in cerebral focal ischemia. J. Pharmacol. Exp. Therap. 296, 312–321. Braiman, L., Alt, A., Kuroki, T., Ohba, M., Bak, A., Tennenbaum, T., Sampson, S.R., 2001. Activation of Protein Kinase Cz induces serine phosphorylation of VAMP2 in the GLUT4 compartment and increases glucose transport in skeletal muscle. Mol. Cell. Biol. 21, 7852–7861. Cazarolli, L.H., Zanatta, L., Jorge, A.P., De Sousa, E., Horst, H., Woehl, V.M., Pizzolatti, M.G., Szpoganicz, B., Silva, F.R.M.B., 2006. Follow-up studies on glycosylated flavonoids and their complexes with vanadium: their anti-hyperglycemic potential role in diabetes. Chem. Biol. Interact. 163, 177–191. Cazarolli, L.H., Zanatta, L., Alberton, E.H., Figueiredo, M.S.R.B., Folador, P., Damazio, R.G., Pizzolatti, M.G., Silva, F.R.M.B., 2008. Flavonoids: cellular and molecular mechanism of action in glucose homeostasis. Mini. Rev. Med. Chem. 8 (10), 1032–1038. Cazarolli, L.H., Folador, P., Moresco, H.H., Brighente, I.M.C., Pizzolatti, M.G., Silva, F.R.M.B., 2009a. Mechanism of action of the stimulatory effect of apigenin-6-C(20 0 -O-alpha-L-rhamnopyranosyl)-beta-L-fucopyranoside on [14C] glucose uptake. Chem. Biol. Interact. 179, 407–412. Cazarolli, L.H., Folador, P., Pizzolatti, M.G., Silva, F.R.M.B., 2009b. Signaling pathways of kaempferol-3-neohesperidoside in glycogen synthesis in rat soleus muscle. Biochimie 91, 843–849. Cazarolli, L.H., Folador, P., Moresco, H.H., Brighente, I.M.C., Pizzolatti, M.G., Silva, F.R.M.B., 2009c. Stimulatory effect of apigenin-6-b-L-fucopyranoside on insulin secretion and glycogen synthesis. Eur. J. Med. Chem. 44, 4668–4673. Chang, L., Chiang, S.H., Saltiel, A.R., 2004. Insulin signaling and the regulation of glucose transport. Mol. Med. 10, 65–71. Chen, Y., Wang, Y., Ji, W., Xu, P., Xu, T., 2008. A pre-docking role for microtubules in insulin-stimulated glucose transporter 4 translocation. FEBS J. 275, 705–712. De Sousa, E., Zanatta, L., Seifriz, I., Creczynski-Pasa, T.B., Pizzolatti, M.G., Szpoganicz, B., Silva, F.R.M.B., 2004. Hypoglycemic effect and antioxidant potential of kaempferol-3,7-O-(alpha)-dirhamnoside from Bauhinia forficata leaves. J. Nat. Prod. 67, 829–832.

Diaz, L.E., Chuan, Y.-C., Lewitt, M., Fernandez-Perez, L., Carrasco-Rodrı´guez, S., Sanchez-Gomez, M., Flores-Morales., A., 2007. IGF-II regulates metastatic properties of choriocarcinoma cells through the activation of the insulin receptor. Mol. Hum. Rep. 13, 567–576. Fang, S.H., Rao, Y.K., Tzeng, Y.M., 2005. Inhibitory effects of flavonol glycosides from Cinnamomum osmophloeum on inflammatory mediators in LPS/IFN-gactivated murine macrophages. Bioorg. Med. Chem. 13, 2381–2388. Fletcher, L.M., Welsh, G.I., Oatey, P.B., Tavare´, J.M., 2000. Role for the microtubule cytoskeleton in GLUT4 vesicle trafficking and in the regulation of insulinstimulated glucose uptake. Biochem. J. 352, 267–276. Folador, P., Cazarolli, L.H., Gazola, A.C., Reginatto, F.H., Schenkel, E.P., Silva, F.R.M.B., 2010. Potential insulin secretagogue effects of isovitexin and swertisin isolated from Wilbrandia ebracteata roots in non-diabetic rats. Fitoterapia 81, 1180–1187. Furtado, L.M., Poon, V., Klip, A., 2003. GLUT4 activation: thoughts on possible mechanism. Acta Physiol. Scand. 178, 287–296. Gu, S.H., Lin, J.L., Lin, P.L., 2010. PTTH-stimulated ERK phosphorylation in prothoracic glands of the silkworm, Bombyx mori: role of Ca2 þ /calmodulin and receptor tyrosine kinase. J. Ins. Physiol. 56, 93–101. Hajduch, E., Litherland, G.J., Hundal, H.S., 2001. Protein kinase B (PKB/Akt) – a key regulator of glucose transport? FEBS Lett. 492, 199–203. Huang, J., Imamura, J.L., Bab, E., Lu, J.C., Olefsky, J.M., 2005. Disruption of microtubules ablates the specificity of insulin signaling to GLUT4 translocation in 3T3-L1 adipocytes. J. Biol. Chem. 280, 42300–42306. Jensen, J., Sharikabad, M.N., Ostbye, K.M., Melien, O., Brors, O., 2003. Evidence that nitroprusside stimulates glucose uptake in isolated rat cardiomyocytes via mitogen-activated protein kinase. Arch. Physiol. Biochem. 111, 239–245. Jorge, A.P., Horst, H., De Sousa, E., Pizzolatti, M.G., Silva, F.R.M.B., 2004. Insulinomimetic effects of kaempferitrin on glycaemia and on [14C] glucose uptake in rat soleus muscle. Chem. Biol. Interact. 149, 89–96. Jung, K.H., Choi, H.S., Kim, D.H., Han, M.Y., Chang, U.J., Yim, S.V., Song, B.C., Kim, C.H., Kang, S.A., 2008. Epigallocatechin gallate stimulates glucose uptake through the phosphatidylinositol 3-kinase-meditaed pathway in L6 rat skeletal muscle cells. J. Med. Food 11, 429–434. Kanzaki, M., Mora, S., Hwang, J.B., Saltiel, A.R., Pessin, J.E., 2004. Atypical protein kinase C (PKC z/l) is a convergent downstream target of the insulinstimulated phosphatidylinositol 3-kinase and TC10 signaling pathways. J. Cell Biol. 164, 279–290. Kanzaki, M., 2006. Insulin receptor signals regulating GLUT4 translocation and actin dynamics. Endocrine. J. 53, 267–293. Klip, A., 2009. The many ways to regulate glucose transporter 4. Appl. Physiol. Nutr. Metab. 34, 481–487. Krisman, C.R., 1962. A method for the colorimetric estimation of glycogen with iodine. Anal. Biochem. 4, 17–23. Krook, A., Wallberg-Henriksson, H., Zierath, J.R., 2004. Sending the signal: molecular mechanisms regulating glucose uptake. Med. Sci. Sports. Exerc., 1212–1217, http://dx.doi.org/10.1249/01.MSS.0000132387.25853.3B. Lane, J., Allan, V., 1998. Microtubule-based membrane movement. Biochim. Biophys. Acta 1376, 27–55. Liu, X.J., He, A.B., Chang, Y.S., Fang, F.D., 2006. Atypical protein kinase C in glucose metabolism. Cell. Sign. 18, 2071–2076. Olson, A.L., Trumbly, A.R., Gibson, G.V., 2001. Insulin-mediated GLUT4 translocation I is dependent on the microtubule network. J. Biol. Chem. 276, 10706–10714. Ong, K.C., Khoo, H.E., 1996. Insulinomimetic effects of myricetin on lipogenesis and glucose transport in rat adipocytes but not glucose transport translocation. Biochem. Pharmacol. 51, 423–429. Ong, K.C., Khoo, H.E., 2000. Effects of myricetin on glycemia and glycogen metabolism in diabetic rats. Life Sci. 67, 1695–1705. Purintrapiban, J., Suttajit, M., Forsberg, N.E., 2006. Differential activation of glucose transport in cultured muscle cells by polyphenolic compounds from Canna indica L. root. Biol. Pharm. Bull. 29, 1995–1998. Salituro, G.M., Pelaez, F., Zhang, B.B., 2001. Discovery of a small molecule insulin receptor activator. Recent Prog. Horm. Res. 56, 107–126. Saperstein, R., Vicario, P.P., Strout, H.V., Brady, E., Slater, E.E., Greenlee, W.J., Ondeyka, D.L., Patchett, A.A., Hangauer, D.G., 1989. Design of a selective insulin receptor tyrosine kinase inhibitor and its effect on glucose uptake and metabolism in intact cells. Biochemistry 28, 5694–5701. Somwar, R., Kim, D.Y., Sweeney, G., Huang, C., Niu, W., Lador, C., Ramlal, T., Klip, A., 2001. GLUT4 translocation precedes the stimulation of glucose uptake by insulin in muscle cells: potential activation of GLUT4 via p38 mitogenactivated protein kinase. Biochem. J. 359, 639–949. Srivastava, A.K., Pandey, S.K., 1998. Potential mechanism(s) involved in the regulation of glycogen synthesis by insulin. Mol. Cell. Biochem. 182, 135–141. Sweeney, G., Somwar, R., Ramlal, T., Volchuk, A., Ueyama, A., Klip, A., 1999. An inhibitor of p38 mitogen-activated protein kinase prevents insulin-stimulated glucose transport but not glucose transporter translocation in 3T3-L1 adipocytes and L6 myotubes. J. Biol. Chem. 274, 10071–10078. Taha, C., Klip, A., 1999. The insulin signaling pathway. J. Membr. Biol. 169, 1–12. Tzeng, Y.M., Chen, K., Rao, Y.K., Lee, M.J., 2009. Kaempferitrin activates the insulin signaling pathway and stimulates secretion of adiponectin in 3T3-L1 adipocytes. Eur. J. Pharmacol. 607, 27–34. Underwood, D.C., Osborn, R.R., Kotzer, C.J., Adams, J.L., Lee, J.C., Webb, E.F., Carpenter, D.C., Bochnowicz, S., Thomas, H.C., Hay, D.W.P., Griswold, D.E., 2000. SB 239063, a potent p38 MAP Kinase inhibitor, reduces inflammatory

Please cite this article as: Cazarolli, L.H., et al., Insulin signaling: A potential signaling pathway for the stimulatory effect of kaempferitrin on glucose uptake in skeletal muscle. Eur J Pharmacol (2013), http://dx.doi.org/10.1016/j.ejphar.2013.02.029i

67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132

L.H. Cazarolli et al. / European Journal of Pharmacology ] (]]]]) ]]]–]]]

1 2 3 4 5 6 7 8 9

cytokine production, airways eosinophil infiltration, and persistence. J. Pharmacol. Exp. Therap. 293, 281–288. Valsa, A.K., Sudheesh, S., Vijayalakshmi, N.R., 1997. Effect of catechin on carbohydrate metabolism. Indian J. Biochem. Biophys. 4, 406–408. Vijayakumar, M.V., Singh, S., Chhipa, R.R., Bhat, M.K., 2005. The hypoglycaemic activity of fenugreek seed extract is mediated through the stimulation of an insulin signalling pathway. Br. J. Pharmacol. 146, 41–48.

7

Watson, R.T., Kanzaki, M., Pessin, J.E., 2004. Regulated membrane trafficking of the insulin-responsive glucose transporter 4 in adipocyte. Endocrine Rev. 25, 177–204. Yamasaki, K., Hishiki, R., Kato, E., Kawabata, J., 2011. Study of kaempferol glycoside as an insulin mimic reveals glycon to be the key active structure. ACS Med. Chem. Lett. 2, 17–21. Zanatta, L., Rosso, A., Folador, P., Figueiredo, M.S.R.B., Pizzolatti, M.G., Leite, L.D., Silva, F.R.M.B., 2008. Insulinomimetic effect of kaempferol 3-neohesperidoside on the rat soleus muscle. J. Nat. Prod. 71, 532–535.

Please cite this article as: Cazarolli, L.H., et al., Insulin signaling: A potential signaling pathway for the stimulatory effect of kaempferitrin on glucose uptake in skeletal muscle. Eur J Pharmacol (2013), http://dx.doi.org/10.1016/j.ejphar.2013.02.029i

10 11 12 13 14 15 16 17