- Email: [email protected]
Cinnamon extract (traditional herb) potentiates in vivo insulin-regulated glucose utilization via enhancing insulin signaling in rats
Diabetes Research and Clinical Practice 62 (2003) 139 /148 www.elsevier.com/locate/diabres
Cinnamon extract (traditional herb) potentiates in vivo insulinregulated glucose utilization via enhancing insulin signaling in rats Bolin Qin a, Masaru Nagasaki b, Ming Ren c, Gustavo Bajotto a, Yoshiharu Oshida a,b, Yuzo Sato a,b,* a
Department of Sports Medicine, Graduate School of Medicine, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan b Research Center of Health, Physical Fitness and Sports, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan c Department of Visual Neuroscience, Graduate School of Medicine, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan Received 12 March 2003; received in revised form 18 June 2003; accepted 17 July 2003
Abstract Cinnamon has been shown to potentiate the insulin effect through upregulation of the glucose uptake in cultured adipocytes. In the present study, we evaluated the effect of the cinnamon extract on the insulin action in awaked rats by the euglycemic clamp and further analyzed possible changes in insulin signaling occurred in skeletal muscle. The rats were divided into saline and cinnamon extract (30 and 300 mg/kg BW-doses: C30 and C300) oral administration groups. After 3-weeks, cinnamon extract treated rats showed a significantly higher glucose infusion rate (GIR) at 3 mU/ kg per min insulin infusions compared with controls (118 and 146% of controls for C30 and C300, respectively). At 30 mU/kg per min insulin infusions, the GIR in C300 rats was increased 17% over controls. There were no significant differences in insulin receptor (IR)-b, IR substrate (IRS)-1, and phosphatidylinositol (PI) 3-kinase protein content between C300 rats and controls. However, the skeletal muscle insulin-stimulated IR-b and the IRS-1 tyrosine phosphorylation levels in C300 rats were 18 and 33% higher, respectively, added to 41% higher IRS-1/PI 3-kinase association. These results suggest that the cinnamon extract would improve insulin action via increasing glucose uptake in vivo, at least in part through enhancing the insulin-signaling pathway in skeletal muscle. # 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Cinnamon extract; Euglycemic clamp; Insulin action; Insulin signaling
1. Introduction Abbreviations: GIR, glucose infusion rate; IR-b, insulin receptor b-subunit; IRS-1, insulin receptor substrate-1; PI 3kinase, phosphatidylinositol 3-kinase. * Corresponding author. Tel.: /81-52-789-3962; fax: /8152-789-3962/3957. E-mail address: [email protected] (Y. Sato).
The cinnamon, also known by Cassia, Sweet Wood, and Gui Zhi, is traditionally harvested in Asian countries. It is, perhaps, one of the oldest herbal medicines, having been mentioned in Chinese texts as long as 4000 years ago [1,2]. The large
0168-8227/03/$ - see front matter # 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/S0168-8227(03)00173-6
140
B. Qin et al. / Diabetes Research and Clinical Practice 62 (2003) 139 /148
number of applications for cinnamon indicates the widespread appreciation that folk herbalists around the world have had for cinnamon as a medicine [2], including the treatment of diarrhea, arthritis, etc. [1]. Furthermore, it has been reported that the cinnamon extract has vasodilative, antithrombotic, antispastic, anti-ulcerous, and antiallergic action [2]. In the last decade, in vitro studies revealed that the cinnamon extract mimics the effect of insulin, which potentiates insulin action in isolated adipocytes [3 /6]. It is believed that the methylhydroxychalcone polymer (MHCP, extracted from cinnamon) is responsible for the above effect. MHCP may be useful in the treatment of insulin resistance via increasing glucose utilization in cells [6]. Moreover, the cinnamon extract has also been shown to improve the insulin receptor function [5,6]. However, to our knowledge, up till now the effect of the cinnamon extract on insulin action has not been demonstrated in in vivo studies. Insulin resistance is a progressive metabolic disorder characterized by reduced glucose uptake in response to normal concentrations of insulin and is most likely the result of a combination of polygenic defects and environmental factors [7 /9]. The resistance to the action of insulin can result from a variety of causes, including defects both in the receptor binding and at the postreceptor levels [10]. Goodyear et al. [11] and Bjornholm et al. [12] demonstrated that significantly less insulin stimulation of its receptor and IRS-1 tyrosine phosphorylation, as well as IRS-1-immunoprecipitable PI 3kinase activity, is detected in skeletal muscle from insulin-resistant subjects compared with controls. To evaluate this hypothesis, in the present study, we first determined whether insulin action was really improved by the cinnamon extract administration to Wistar rats by the euglycemic clamp technique. Secondly, as the skeletal muscle is the principal tissue responsible for the insulin-stimulated glucose disposal [13] and the major site of peripheral insulin resistance [14], and due to previous reports that the cinnamon extract affects elements of the PI 3-kinase upstream and activates the insulin receptor kinase [5], we further analyzed whether the cinnamon extract could enhance
insulin signaling in skeletal muscle harvested from the experimental animals.
2. Materials and methods 2.1. Animals and materials Eighteen male Wistar rats, aged 6 weeks, weighing between 145 and 160 g were purchased form CLEA (Japan). Neutral insulin was purchased from Novo Nordisk (Denmark). Antiphosphatidylinositol 3-kinase (anti-PI 3-kinase), anti-insulin receptor b-subunit (anti-IR-b) and anti-insulin receptor substrate-1 (anti-IRS-1) antibodies were obtained from Santa Cruz Biotechnology (USA). Anti-phosphotyrosine antibody was purchased from Upstate Biotechnology (USA). All other reagents were of biochemical grade. The herbal extract of cinnamon was kindly provided by Tsumura Co. (Japan). Cinnamon was extracted with hot water, filtered, lyophilized, and then stored at 4 8C. 2.2. Experimental design Animals were housed in individual cages in a room with controlled temperature (23 8C) and light (12-h light:12-h dark cycle; lighting between 08:00 and 20:00 h) and had free access to a standard diet and water. All procedures were in accordance with the Guide for the Care and Use of Laboratory Animals of Nagoya University. After a 1-week acclimation period, the rats were randomly divided into three groups: saline group, 30 mg/kg BW cinnamon extract treatment group, and 300 mg/kg BW cinnamon extract treatment group. The herbal extract was dissolved with saline, and oral saline or cinnamon extract loading was carried out daily for 3 weeks. At the end of the second week, the rats were submitted to the surgery procedures. 2.3. Surgery procedures The animals were anesthetized with an intraperitoneal injection of 50 mg/kg BW sodium pentobarbital. Thereafter, a middle ventral incision was
B. Qin et al. / Diabetes Research and Clinical Practice 62 (2003) 139 /148
made in the neck and the right jugular vein and left carotid artery were cannulated with Silascon SH tubing (Osaka Japan). The catheters were tunneled subcutaneously to the dorsal region of the neck and flushed with 300 ml of saline containing heparin (40 U/ml) and 500 ml of sodium penicillin G (10 000 U/ml). The catheters were then filled with a viscous solution of 50% polyvinylpyrrolidone (PVP) and capped with a piece of polyethylene tubing melted and sealed at one end. After surgery, the rats were kept at the same preoperative conditions. The food intake was recovered 2/ 3 days after surgery. 2.4. Euglycemic clamp procedures One week after surgery, each rat was submitted to a two-step hyperinsulinemic euglycemic clamp procedure, after an overnight fast to assess the whole-body insulin action. The rat was placed in a restraining cage and extension tubing was attached to the jugular catheter by an adapter for the continuous infusion of insulin and glucose. The carotid catheter was used for blood sampling. A primed insulin infusion was delivered at a rate of 3 mU/kg per min (low-dose) for 90 min and then at an increased rate of 30 mU/kg per min (maximal stimulation, high-dose) for an additional 90 min period. Based on the blood glucose concentration measured every 10 min, the blood glucose was kept constant at the basal level with a variable infusion of a 20% (w/v) glucose solution. Additional blood samples were collected just before insulin infusions and at 90 and 180 min after starting insulin infusion for the determination of the plasma insulin concentrations. The glucose infusion rate (GIR) in mg/kg per min was calculated every 10 min during the clamp study period. The means of GIR values from 60 to 90 min and from 150 to 180 min for the two-step sequential euglycemic clamp procedure were regarded as an index of the whole body insulin action since a plateau in the GIR was achieved during these periods of time, as reported previously [15 /17]. At the end of the euglycemic clamp, skeletal muscle was excised, made free of blood, and immediately frozen at liquid nitrogen temperature before it was stored at /80 8C until analysis.
141
2.5. Blood assays Blood glucose concentration was determined using a glucose analyzer (model 2300A; Yellow Springs Instrument, OH). Plasma insulin was assayed with a radioimmunoassay kit (Phadesepa Insulin RIA, Pharmacia AB, Sweden). Plasma free fatty acids (FFA) were determined with a commercial kit (Wako pure Chemical Industries, Osaka, Japan).
2.6. Tissue processing Muscle samples were homogenized in ice-cold homogenizing buffer (20 mM Tris /Cl, pH 7.6, 150 mM NaCl, 1% Nonidet P-40, 10 mM NaF, 10 mM Na3VO4, 10 mM EDTA, 1 mM PMSF, 1 mg/ml Leupeptin, and 1 mg/ml Aprotinin) using a Polytron homogenizer as described by [18]. The homogenates were kept at ice temperature for 1 h and then centrifuged at 38 000 rpm (150 000 /g ) at 4 8C for 1 h using a Hitachi RP55 rotor (Hitachi, Tokyo, Japan), the protein concentration of the supernatants was determined using a commercial kit (Bio-Rad, Richmond, CA). Supernatant was stored at /80 8C until used.
2.7. Protein expression For the assays of the protein expression of the IR-b, IRS-1, and PI3-kinase, an aliquot (40 mg) of the supernatant was resuspended in treatment buffer containing b-mercaptoethanol and boiled for 5 min. The supernatant proteins (40 mg) were size-fractionated by SDS-PAGE (6 or 10% acrylamide gels). After electroblotting, the PVDF membranes were incubated overnight with the anti-IR-b, anti-IRS-1, or anti-PI3-kinase antibodies at 4 8C. The membrane was then incubated with Goat Anti-Rabbit IgG (1:2000) in Tris / buffer (pH 7.4) containing 3% BSA for 1 h at room temperature. After washing, blotted proteins were visualized using a Western blotting detection system (ECL Plus, Amersham, UK). Quantification of the band intensity was performed using the public domain NIH image software.
142
B. Qin et al. / Diabetes Research and Clinical Practice 62 (2003) 139 /148
2.8. Insulin receptor-b and IRS-1 tyrosine phosphorylation and IRS-1 associated with PI 3kinase The supernatants containing equal amounts of protein (1 mg/ml each tube) were incubated overnight with anti-IR-b (5 mg/ml) or anti-IRS-1 (5 mg/ml) at 4 8C, as indicated, and then with 20 ml of protein A agarose beads at 4 8C for 4 h. The immune complexes were washed as described by [11]. Samples were resuspended in treatment buffer containing b-mercaptoethanol and boiled for 5 min. Phosphorylated proteins were separated by SDS-PAGE (6 or 10% acrylamide gels). After electroblotting, the PVDF membranes were incubated with phosphotyrosine antibody or PI3kinase antibody. Phosphorylated proteins were visualized by the same method mentioned above. 2.9. Statistical analysis All data are expressed as mean9/S.E. Data were analyzed by the one-way analysis of variance. When a significant difference was found (P B/ 0.05), the results were further compared with the Fisher’s PLSD test. The StatView 5.0 software (SAS Institute Inc., Cary, NC) was used for the statistical analysis.
3. Results 3.1. Body weight, plasma FFA, blood glucose, and plasma insulin levels The body weight, plasma FFA, blood glucose, and plasma insulin levels before, during and immediately after the euglycemic clamp are shown in Table 1. The final body weights of the cinnamon treated rats and the controls were not significantly different. Although body weights were decreased in all rats after surgery for cannulation, they returned to preoperative levels after 3 days. Cinnamon treatment for 3 weeks did not affect plasma FFA and fasting blood glucose concentrations in either 30 or 300-mg/kg dose treated rats. Basal and steady state plasma insulin concentrations during the low-dose and the high-dose
insulin clamp procedures were not significantly different among all experimental groups; namely, 3-weeks cinnamon extract administration did not affect insulin secretion. 3.2. Effect of cinnamon extract administration on GIRs The average GIRs for the last 30 min during the low-dose and high-dose insulin infusion rates are shown in Figs. 1 and 2. Because a plateau GIR was achieved, GIR was used as an indicator of wholebody glucose utilization. At the low-dose insulin clamp studies, the GIRs of the cinnamon extractadministered groups were significantly increased (30 mg/kg dose: 118% of controls, P B/0.05; 300 mg/kg dose: 146% of controls, P B/0.001), as shown in Fig. 2. In the high-dose insulin infusion, the GIR of the rats treated with a 300-mg/kg dose of cinnamon extract was also significantly greater than that of controls (117% of controls, P B/0.05). However, a 30-mg/kg dose did not affect the GIR at the high-dose clamp. 3.3. Effect of cinnamon extract administration on IR-b, IRS-1, and PI 3-kinase protein contents in rat skeletal muscle The total protein content of IR-b, IRS-1, and PI 3-kinase was measured in skeletal muscles of all rats. No significant difference in the protein content of IR-b, IRS-1, and PI 3-kinase was detected (Fig. 3A, Fig. 4A, Fig. 5A, respectively). 3.4. Effect of cinnamon extract administration on tyrosine-phosphorylation of IR-b and IRS-1, and the IRS-1 association with PI 3-kinase in rat skeletal muscle The tyrosine phosphorylation level of IR-b was determined by immunoblotting the phosphotyrosine antibody immunoprecipitates with the IR-b antibody. As shown in Fig. 3B, the tyrosine phosphorylation level of IR-b in skeletal muscle of the cinnamon extract administered animals was significantly increased when compared with controls (118% of control, P B/0.05). The same tendency was found for the IRS-1 tyrosine phos-
B. Qin et al. / Diabetes Research and Clinical Practice 62 (2003) 139 /148
143
Table 1 Body weight, plasma FFA, and concentrations of blood glucose and plasma insulin before and immediately after the euglycemic clamp procedure at low-dose and high-dose insulin infusion Group
Saline Cinnamon (30 mg/kg BW) Cinnamon (300 mg/kg BW)
Body weight (g)
2719/3 2759/4 2729/5
FFA (mEq/l)
0.649/0.04 0.669/0.05 0.689/0.06
phorylation (133% of controls, P B/0.01), as shown in Fig. 4B. The IRS-1/PI 3-kinase association level was determined by immunoblotting the PI 3-kinase antibody immunoprecipitates with the IRS-1 antibody. As shown in Fig. 5B, the IRS-1/PI 3-kinase association had a significant increase in the skeletal muscles of cinnamon extract treated animals (41% above controls, P B/0.01).
4. Discussion In vitro studies have suggested that the cinnamon extract acts as an insulin mimetic, enhancing the glucose uptake in adipocytes [3 /6]. Addition-
Glucose (mg/dl)
Insulin (mU/ml)
Basal
3.0
30.0
Basal
3.0
30.0
759/2 739/3 719/3
749/3 749/2 759/2
739/1 739/4 729/3
7.89/0.1 8.09/0.3 7.99/0.2
299/3 319/2 309/2
5809/15 6129/19 6059/25
ally, treatment with cinnamon extract has been reported to enhance IR kinase activity, autophosphorylation of IR, glycogen synthesis, and glycogen synthase activity in 3T3-L1 adipocytes [6]. The present study was undertaken to assess the effect of different doses of cinnamon extract on the insulin action in vivo in the conscious unstressed rats. We investigated the insulin-induced glucose uptake using a two-step hyperinsulinemic euglycemic clamp procedure. Researches on the wholebody insulin-mediated glucose utilization under conditions of euglycemic hyperinsulinemia suggested that the net splanchnic glucose uptake is similar in control and type 2 diabetes mellitus subjects; in addition, adipose tissue glucose uptake
Fig. 1. GIRs for the euglycemic clamp procedure period at the low-dose (3.0 mU/kg per min) and the high-dose (30.0 mU/kg per min) insulin infusion for animals treated with saline, 30 and 300-mg/kg cinnamon extract dose. (m) Saline treated; (") 30-mg/kg dose cinnamon extract treated; (k) 300-mg/kg dose cinnamon extract treated. Values are mean9/S.E.
144
B. Qin et al. / Diabetes Research and Clinical Practice 62 (2003) 139 /148
Fig. 2. GIR during the euglycemic clamp procedure in normal control and cinnamon treated (30 and 300-mg/kg BW, respectively), rats at the low-dose (3.0 mU/kg per min) and high-dose (30.0 mU/kg per min) insulin infusion. Data are expressed as the mean9/S.E. for six rats in each group. *, P B/0.05 vs. control; **, P B/0.001 vs. control.
is assumed to represent /2% of the total glucose disposal [19,20]. Muscle glucose uptake in control subjects represents /75% of the total glucose metabolism [13,14,21] and in type 2 diabetic subjects accounts for essentially all of the impairment in insulin-mediated glucose uptake. In other words, the insulin-stimulated glucose disposal during the euglycemic clamp is mainly credited to the glucose uptake in skeletal muscle. During this study, a high physiological insulin level of
about 30 mU/ml achieved during low-dose insulin infusion rate reflects insulin sensitivity in peripheral tissues (i.e. skeletal muscle) and liver in the postprandial state. Changes in GIR are thought to be caused mainly by changes in the insulin receptor binding at high physiological insulin levels. Although we did not measure the effect of insulin on hepatic glucose production (HGP), according to Tominaga et al. [22], HGP can be completely suppressed under an insulin infusion
Fig. 3. Effect of 3-weeks 300-mg/kg BW dose cinnamon extract administration on IR-b protein content (A) and IR-b tyrosine phosphorylation (B) in gastrocnemius of rats. Muscle proteins were resolved by 10% SDS-PAGE, and tyrosine phosphorylation was detected by immunoprecipitation with anti-IR- antibody followed by immunoblot analysis with antiphosphotyrosine antibody. Data are expressed as the mean9/S.E. for five rats in each group. *, P B/0.05 vs. controls.
B. Qin et al. / Diabetes Research and Clinical Practice 62 (2003) 139 /148
145
Fig. 4. Effect of 3-weeks 300-mg/kg BW dose cinnamon extract administration on IRS-1 protein content (A) and IRS-1 tyrosine phosphorylation (B) in gastrocnemius of rats. Muscle proteins were resolved by 6% SDS-PAGE, and tyrosine phosphorylation was detected by immunoprecipitation with anti-IRS-1 antibody followed by immunoblot analysis with antiphosphotyrosine antibody. Data are expressed as the mean9/S.E. for five rats in each group. *, P B/0.01 vs. controls.
Fig. 5. Effect of 3-weeks 300-mg/kg BW dose cinnamon extract administration on PI 3-kinase protein content (A) and IRS-1/PI 3kinase association (B) in gastrocnemius of rats. Muscle proteins were resolved by 10% SDS-PAGE, and IRS-1/PI 3-kinase association was detected by immunoprecipitation with anti-IRS-1 antibody followed by immunoblot analysis with anti-PI 3-kinase antibody. Data are expressed as the mean9/S.E. for five rats in each group. *, P B/0.01 vs. controls.
146
B. Qin et al. / Diabetes Research and Clinical Practice 62 (2003) 139 /148
rate of 3 mU/kg per min, and an even lower infusion rate (2.4 mU/kg per min) was shown to suppress around 90% of the HGP in conscious unstressed rats [23], therefore, the GIR is essentially synonymous with the rate of the total body glucose utilization [23]. Naturally, these effects of insulin infusion on HGP or its action on peripheral tissues are passive of change according to the experimental conditions (i.e. type of animal, sex, age, diet, anesthesia or awake condition, and stress), for example, Lam et al. [24] reported that 5 mU/kg per min of insulin infusion suppressed endogenous glucose production (EGP) by /55% only. EGP was inhibited by insulin in a dosedependent fashion [23], the high-dose insulin infusion leads to a maximal insulin action and insulin responsiveness, predominantly indicating the capacity of post-receptor binding mechanisms [25,26], and resulted in the pharmacological insulin range, which sufficiently suppresses HGP [23]. Therefore, the results of the present study suggest that cinnamon extract administration would improve the insulin sensitivity and responsiveness in peripheral tissues in a dose-dependent manner (Figs. 1 and 2). The insulin signaling pathway leading to the cellular uptake of glucose begins with the binding of insulin to its receptor (IR); in the sequence, ligand binding to the IR induces autophosphorylation of specific tyrosine residues, which then activates the substrate protein tyrosine kinase of the IR [27]. The activated IR phosphorylates several intracellular proteins, including the IRS1. Tyrosine phosphorylation of the IRS-1 leads to the binding of PI 3-kinase [28] and the activation of its enzymatic activity, a necessary step for the translocation of glucose transporter-4 to the plasma membrane, consequently elevating the rate of cellular glucose uptake in response to insulin [29]. Reduced IRS-1 expression was observed in Zucker fatty rats [30], and mice that are deficient in the IRS-1, prepared by target geneknockout, exhibited hyperinsulinemia and glucose intolerance [31 /33]. These findings propose that decreased IRS-1 protein expression is involved in the development of insulin resistance. On the other hand, several defects in insulin signaling have been reported in type 2 diabetes, including a modest
reduction in insulin receptor phosphorylation and tyrosine kinase activity [34], decrease in insulinstimulated IRS-1 tyrosine phosphorylation [35,36], and reduced PI 3-kinase activity [35 /37]. However, the IRS-1 protein expression appears to be unchanged in these studies [35 /37]. Regardless of differences between humans and animals or the progress of specific diseases, the decrease in insulin effect is unquestionably caused by changes in insulin signaling (protein expression and/or phosphorylation level). Therefore, we further clarified the effect of cinnamon extract treatment on the molecular mechanisms of the in vivo insulin signaling in skeletal muscle obtained from the experimental rats. Our data show that cinnamon extract treatment does not affect the IRS-1 protein amount (Fig. 4A), but significantly enhances the tyrosine phosphorylation level of IRS-1 (Fig. 4B). Additionally, the same trend was found in the IRb, the metabolic upstream effects of IRS-1 (Fig. 3B), in good agreement with the result reported by Imparl-Radosevich et al. [5]. The activation of PI 3-kinase is required for insulin and stimulates the glucose transport [38,39]. Although we have not assessed directly the effect on the activation of PI 3-kinase, it was previously described that, in parallel with an increase in IRS-1/PI 3-kinase association in skeletal muscle of STZ-diabetes [40], there is an increase in insulin-stimulated IRS-1 tyrosine phosphorylation and IRS-1-associated PI 3-kinase activity [40]. To be precise, PI 3-kinase activation closely correlates with IRS-1 phosphorylation [40]. The present data revealed that cinnamon extract treatment increases IRS-1 tyrosine phosphorylation levels and the IRS-1/PI 3-kinase association (Fig. 5B); therefore, these changes are thought to result in improved activation of PI 3-kinase. In summary, the present study showed for the first time that (1) oral treatment with the cinnamon extract enhances the glucose utilization in vivo, and this effect occurs in a dose-dependent manner; (2) the cinnamon extract potentiates the insulin-stimulated tyrosine phosphorylation of IRb and IRS-1 and the IRS-1 association with PI 3kinase, however, without affecting their protein content levels.
B. Qin et al. / Diabetes Research and Clinical Practice 62 (2003) 139 /148
In conclusion, the current study suggests that the oral treatment with cinnamon extract would improve in vivo insulin-regulated whole-body glucose utilization in a dose-dependent fashion in rats, at least in part through enhancing the insulin signaling in skeletal muscle. Further investigation on the effect of the cinnamon extract on the adultonset of diabetes is needed.
Acknowledgements This work was supported in part by Grants of the Research from Projects on Aging and Health from the Ministry of Health, Welfare and Labor of Japan (H13-009) and from the Kampo Science Foundation of Japan (2002).
References [1] F.S. Leung, A.Y., in: Encyclopedia of common natural ingredients used in foods, drugs, and cosmetics, second ed., Wiley, New York, 1996, pp. 168 /170. [2] K. Toriizuka, Basic lecture of Kampo medicine: pharmacological effect of cinnamon, Kampo Med. 11 (1998) 431 / 436. [3] C.L. Broadhurst, M.M. Polansky, R.A. Anderson, Insulinlike biological activity of culinary and medicinal plant aqueous extracts in vitro, J. Agric. Food Chem. 48 (2000) 849 /852. [4] A. Khan, N.A. Bryden, M.M. Polansky, R.A. Anderson, Insulin potentiating factor and chromium content of selected foods and spices, Biol. Trace Elem. Res. 24 (1990) 183 /188. [5] J. Imparl-Radosevich, S. Deas, M.M. Polansky, D.A. Baedke, T.S. Ingebritsen, R.A. Anderson, D.J. Graves, Regulation of PTP-1 and insulin receptor kinase by fractions from cinnamon: implications for cinnamon regulation of insulin signalling, Horm. Res. 50 (1998) 177 /182. [6] K.J. Jarvill-Taylor, R.A. Anderson, D.J. Graves, A hydroxychalcone derived from cinnamon functions as a mimetic for insulin in 3T3-L1 adipocytes, J. Am. Coll. Nutr. 20 (2001) 327 /336. [7] M.P. Czech, Molecular actions of insulin on glucose transport, Annu. Rev. Nutr. 15 (1995) 441 /471. [8] G.M. Reaven, Pathophysiology of insulin resistance in human disease, Physiol. Rev. 75 (1995) 473 /486. [9] C.R. Kahn, Banting lecture. Insulin action, diabetogenes, and the cause of type 2 diabetes, Diabetes 43 (1994) 1066 / 1084.
147
[10] J.M. Olefsky, O.G. Kolterman, J.A. Scarlett, Insulin action and resistance in obesity and noninsulin-dependent type 2 diabetes mellitus, Am. J. Physiol. 243 (1982) E15 / E30. [11] L.J. Goodyear, F. Giorgino, L.A. Sherman, J. Carey, R.J. Smith, G.L. Dohm, Insulin receptor phosphorylation, insulin receptor substrate-1 phosphorylation, and phosphatidylinositol 3-kinase activity are decreased in intact skeletal muscle strips from obese subjects, J. Clin. Invest. 95 (1995) 2195 /2204. [12] M. Bjornholm, Y. Kawano, M. Lehtihet, J.R. Zierath, Insulin receptor substrate-1 phosphorylation and phosphatidylinositol 3-kinase activity in skeletal muscle from NIDDM subjects after in vivo insulin stimulation, Diabetes 46 (1997) 524 /527. [13] R.A. DeFronzo, E. Jacot, E. Jequier, E. Maeder, J. Wahren, J.P. Felber, The effect of insulin on the disposal of intravenous glucose. Results from indirect calorimetry and hepatic and femoral venous catheterization, Diabetes 30 (1981) 1000 /1007. [14] R.A. DeFronzo, R. Gunnarsson, O. Bjorkman, M. Olsson, J. Wahren, Effects of insulin on peripheral and splanchnic glucose metabolism in noninsulin-dependent (type 2) diabetes mellitus, J. Clin. Invest. 76 (1985) 149 /155. [15] L. Li, Y. Oshida, M. Kusunoki, K. Yamanouchi, B.L. Johansson, J. Wahren, Y. Sato, Rat C peptide I and II stimulate glucose utilization in STZ-induced diabetic rats, Diabetologia 42 (1999) 958 /964. [16] Y. Oshida, Y. Tachi, Y. Morishita, K. Kitakoshi, N. Fuku, Y.Q. Han, I. Ohsawa, Y. Sato, Nitric oxide decreases insulin resistance induced by high-fructose feeding, Horm. Metab. Res. 32 (2000) 339 /342. [17] X. Hu, J. Sato, Y. Oshida, M. Xu, G. Bajotto, Y. Sato, Effect of Gosha-jinki-gan (chinese herbal medicine: NiuChe-Sen-Qi-Wan) on insulin resistance in streptozotocininduced diabetic rats, Diabetes Res. Clin. Pract. 59 (2003) 103 /111. [18] M. Nagasaki, N. Nakai, Y. Oshida, Z. Li, M. Xu, M. Obayashi, T. Murakami, A. Yoshimura, N. Fujitsuka, Y. Shimomura, Y. Sato, Exercise training prevents maturation-induced decreases in insulin receptor substrate-1 and phosphatidylinositol 3-kinase in rat skeletal muscle, Metabolism 49 (2000) 954 /959. [19] P. Bjorntorp, L. Sjostrom, Carbohydrate storage in man: speculations and some quantitative considerations, Metabolism 27 (1978) 1853 /1865. [20] P. Marin, M. Rebuffe-Scrive, U. Smith, P. Bjorntorp, Glucose uptake in human adipose tissue, Metabolism 36 (1987) 1154 /1160. [21] R.A. DeFronzo, Lilly lecture, The triumvirate: beta-cell, muscle, liver. A collusion responsible for NIDDM, Diabetes 37 (1988) (1987) 667 /687. [22] M. Tominaga, M. Kimura, K. Sugiyama, T. Abe, K. Igarashi, M. Igarashi, H. Eguchi, A. Sekikawa, A. Ogawa, H. Manaka, et al., Effects of seishin-renshi-in and Gymnema sylvestre on insulin resistance in streptozotocin-
148
[23]
[24]
[25]
[26]
[27]
[28]
[29]
[30]
[31]
[32]
B. Qin et al. / Diabetes Research and Clinical Practice 62 (2003) 139 /148 induced diabetic rats, Diabetes Res. Clin. Pract. 29 (1993) 11 /17. D. Smith, L. Rossetti, E. Ferrannini, C.M. Johnson, C. Cobelli, G. Toffolo, L.D. Katz, R.A. DeFronzo, In vivo glucose metabolism in the awake rat: tracer and insulin clamp studies, Metabolism 36 (1987) 1167 /1174. T.K. Lam, H. Yoshii, C.A. Haber, E. Bogdanovic, L. Lam, I.G. Fantus, A. Giacca, Free fatty acid-induced hepatic insulin resistance: a potential role for protein kinase C-delta, Am. J. Physiol. Endocrinol. Metab. 283 (2002) E682 /E691. C.R. Khan, Insulin resistance, insulin insensitivity and insulin unresponsiveness: a necessary distinction, Metabolism 27 (1978) 1893 /1902. J.M. Olefsky, O.G. Kolterman, J.A. Scarlett, Insulin action and resistance in obesity and noninsulin-dependent type 2 diabetes mellitus, Am. J. Physiol. 243 (1999) E15 / E30. C.R. Kahn, M.F. White, The insulin receptor and the molecular mechanism of insulin action, J. Clin. Invest. 82 (1988) 1151 /1156. M.F. White, The IRS-signalling system: a network of docking proteins that mediate insulin action, Mol. Cell. Biochem. 182 (1998) 3 /11. K. Hara, K. Yonezawa, H. Sakaue, A. Ando, K. Kotani, T. Kitamura, Y. Kitamura, H. Ueda, L. Stephens, T.R. Jackson, et al., 1-Phosphatidylinositol 3-kinase activity is required for insulin-stimulated glucose transport but not for RAS activation in CHO cells, Proc. Natl. Acad. Sci. USA 91 (1994) 7415 /7419. M. Anai, M. Funaki, T. Ogihara, J. Terasaki, K. Inukai, H. Katagiri, Y. Fukushima, Y. Yazaki, M. Kikuchi, Y. Oka, T. Asano, Altered expression levels and impaired steps in the pathway to phosphatidylinositol 3-kinase activation via insulin receptor substrates 1 and 2 in Zucker fatty rats, Diabetes 47 (1998) 13 /23. E. Araki, M.A. Lipes, M.E. Patti, J.C. Bruning, B.R. Haag, R.S. Johnson, C.R. Kahn, Alternative pathway of insulin signalling in mice with targeted disruption of the IRS-1 gene, Nature 372 (1994) 186 /190. H. Tamemoto, T. Kadowaki, K. Tobe, T. Yagi, H. Sakura, T. Hayakawa, Y. Terauchi, K. Ueki, Y. Kaburagi, S. Satoh, et al., Insulin resistance and growth retardation in mice lacking insulin receptor substrate-1, Nature 372 (1994) 182 /186.
[33] T. Yamauchi, K. Tobe, H. Tamemoto, K. Ueki, Y. Kaburagi, R. Yamamoto-Honda, Y. Takahashi, F. Yoshizawa, S. Aizawa, Y. Akanuma, N. Sonenberg, Y. Yazaki, T. Kadowaki, Insulin signalling and insulin actions in the muscles and livers of insulin-resistant, insulin receptor substrate 1-deficient mice, Mol. Cell. Biol. 16 (1996) 3074 / 3084. [34] A. Handberg, A. Vaag, J. Vinten, H. Beck-Nielsen, Decreased tyrosine kinase activity in partially purified insulin receptors from muscle of young, non-obese first degree relatives of patients with type 2 (non-insulindependent) diabetes mellitus, Diabetologia 36 (1993) 668 /674. [35] W. Pratipanawatr, T. Pratipanawatr, K. Cusi, R. Berria, J.M. Adams, C.P. Jenkinson, K. Maezono, R.A. DeFronzo, L.J. Mandarino, Skeletal muscle insulin resistance in normoglycemic subjects with a strong family history of type 2 diabetes is associated with decreased insulinstimulated insulin receptor substrate-1 tyrosine phosphorylation, Diabetes 50 (2001) 2572 /2578. [36] H. Storgaard, X.M. Song, C.B. Jensen, S. Madsbad, M. Bjornholm, A. Vaag, J.R. Zierath, Insulin signal transduction in skeletal muscle from glucose-intolerant relatives of type 2 diabetic patients [corrected], Diabetes 50 (2001) 2770 /2778. [37] K. Cusi, K. Maezono, A. Osman, M. Pendergrass, M.E. Patti, T. Pratipanawatr, R.A. DeFronzo, C.R. Kahn, L.J. Mandarino, Insulin resistance differentially affects the PI 3-kinase- and MAP kinase-mediated signaling in human muscle, J. Clin. Invest. 105 (2000) 311 /320. [38] B. Cheatham, C.J. Vlahos, L. Cheatham, L. Wang, J. Blenis, C.R. Kahn, Phosphatidylinositol 3-kinase activation is required for insulin stimulation of pp70 S6 kinase, DNA synthesis, and glucose transporter translocation, Mol. Cell. Biol. 14 (1994) 4902 /4911. [39] T. Okada, Y. Kawano, T. Sakakibara, O. Hazeki, M. Ui, Essential role of phosphatidylinositol 3-kinase in insulininduced glucose transport and antilipolysis in rat adipocytes. Studies with a selective inhibitor wortmannin, J. Biol. Chem. 269 (1994) 3568 /3573. [40] F. Folli, M.J. Saad, J.M. Backer, C.R. Kahn, Regulation of phosphatidylinositol 3-kinase activity in liver and muscle of animal models of insulin-resistant and insulindeficient diabetes mellitus, J. Clin. Invest. 92 (1993) 1787 / 1794.
Cinnamon extract (traditional herb) potentiates in vivo insulinregulated glucose utilization via enhancing insulin signaling in rats Bolin Qin a, Masaru Nagasaki b, Ming Ren c, Gustavo Bajotto a, Yoshiharu Oshida a,b, Yuzo Sato a,b,* a
Department of Sports Medicine, Graduate School of Medicine, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan b Research Center of Health, Physical Fitness and Sports, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan c Department of Visual Neuroscience, Graduate School of Medicine, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan Received 12 March 2003; received in revised form 18 June 2003; accepted 17 July 2003
Abstract Cinnamon has been shown to potentiate the insulin effect through upregulation of the glucose uptake in cultured adipocytes. In the present study, we evaluated the effect of the cinnamon extract on the insulin action in awaked rats by the euglycemic clamp and further analyzed possible changes in insulin signaling occurred in skeletal muscle. The rats were divided into saline and cinnamon extract (30 and 300 mg/kg BW-doses: C30 and C300) oral administration groups. After 3-weeks, cinnamon extract treated rats showed a significantly higher glucose infusion rate (GIR) at 3 mU/ kg per min insulin infusions compared with controls (118 and 146% of controls for C30 and C300, respectively). At 30 mU/kg per min insulin infusions, the GIR in C300 rats was increased 17% over controls. There were no significant differences in insulin receptor (IR)-b, IR substrate (IRS)-1, and phosphatidylinositol (PI) 3-kinase protein content between C300 rats and controls. However, the skeletal muscle insulin-stimulated IR-b and the IRS-1 tyrosine phosphorylation levels in C300 rats were 18 and 33% higher, respectively, added to 41% higher IRS-1/PI 3-kinase association. These results suggest that the cinnamon extract would improve insulin action via increasing glucose uptake in vivo, at least in part through enhancing the insulin-signaling pathway in skeletal muscle. # 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Cinnamon extract; Euglycemic clamp; Insulin action; Insulin signaling
1. Introduction Abbreviations: GIR, glucose infusion rate; IR-b, insulin receptor b-subunit; IRS-1, insulin receptor substrate-1; PI 3kinase, phosphatidylinositol 3-kinase. * Corresponding author. Tel.: /81-52-789-3962; fax: /8152-789-3962/3957. E-mail address: [email protected] (Y. Sato).
The cinnamon, also known by Cassia, Sweet Wood, and Gui Zhi, is traditionally harvested in Asian countries. It is, perhaps, one of the oldest herbal medicines, having been mentioned in Chinese texts as long as 4000 years ago [1,2]. The large
0168-8227/03/$ - see front matter # 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/S0168-8227(03)00173-6
140
B. Qin et al. / Diabetes Research and Clinical Practice 62 (2003) 139 /148
number of applications for cinnamon indicates the widespread appreciation that folk herbalists around the world have had for cinnamon as a medicine [2], including the treatment of diarrhea, arthritis, etc. [1]. Furthermore, it has been reported that the cinnamon extract has vasodilative, antithrombotic, antispastic, anti-ulcerous, and antiallergic action [2]. In the last decade, in vitro studies revealed that the cinnamon extract mimics the effect of insulin, which potentiates insulin action in isolated adipocytes [3 /6]. It is believed that the methylhydroxychalcone polymer (MHCP, extracted from cinnamon) is responsible for the above effect. MHCP may be useful in the treatment of insulin resistance via increasing glucose utilization in cells [6]. Moreover, the cinnamon extract has also been shown to improve the insulin receptor function [5,6]. However, to our knowledge, up till now the effect of the cinnamon extract on insulin action has not been demonstrated in in vivo studies. Insulin resistance is a progressive metabolic disorder characterized by reduced glucose uptake in response to normal concentrations of insulin and is most likely the result of a combination of polygenic defects and environmental factors [7 /9]. The resistance to the action of insulin can result from a variety of causes, including defects both in the receptor binding and at the postreceptor levels [10]. Goodyear et al. [11] and Bjornholm et al. [12] demonstrated that significantly less insulin stimulation of its receptor and IRS-1 tyrosine phosphorylation, as well as IRS-1-immunoprecipitable PI 3kinase activity, is detected in skeletal muscle from insulin-resistant subjects compared with controls. To evaluate this hypothesis, in the present study, we first determined whether insulin action was really improved by the cinnamon extract administration to Wistar rats by the euglycemic clamp technique. Secondly, as the skeletal muscle is the principal tissue responsible for the insulin-stimulated glucose disposal [13] and the major site of peripheral insulin resistance [14], and due to previous reports that the cinnamon extract affects elements of the PI 3-kinase upstream and activates the insulin receptor kinase [5], we further analyzed whether the cinnamon extract could enhance
insulin signaling in skeletal muscle harvested from the experimental animals.
2. Materials and methods 2.1. Animals and materials Eighteen male Wistar rats, aged 6 weeks, weighing between 145 and 160 g were purchased form CLEA (Japan). Neutral insulin was purchased from Novo Nordisk (Denmark). Antiphosphatidylinositol 3-kinase (anti-PI 3-kinase), anti-insulin receptor b-subunit (anti-IR-b) and anti-insulin receptor substrate-1 (anti-IRS-1) antibodies were obtained from Santa Cruz Biotechnology (USA). Anti-phosphotyrosine antibody was purchased from Upstate Biotechnology (USA). All other reagents were of biochemical grade. The herbal extract of cinnamon was kindly provided by Tsumura Co. (Japan). Cinnamon was extracted with hot water, filtered, lyophilized, and then stored at 4 8C. 2.2. Experimental design Animals were housed in individual cages in a room with controlled temperature (23 8C) and light (12-h light:12-h dark cycle; lighting between 08:00 and 20:00 h) and had free access to a standard diet and water. All procedures were in accordance with the Guide for the Care and Use of Laboratory Animals of Nagoya University. After a 1-week acclimation period, the rats were randomly divided into three groups: saline group, 30 mg/kg BW cinnamon extract treatment group, and 300 mg/kg BW cinnamon extract treatment group. The herbal extract was dissolved with saline, and oral saline or cinnamon extract loading was carried out daily for 3 weeks. At the end of the second week, the rats were submitted to the surgery procedures. 2.3. Surgery procedures The animals were anesthetized with an intraperitoneal injection of 50 mg/kg BW sodium pentobarbital. Thereafter, a middle ventral incision was
B. Qin et al. / Diabetes Research and Clinical Practice 62 (2003) 139 /148
made in the neck and the right jugular vein and left carotid artery were cannulated with Silascon SH tubing (Osaka Japan). The catheters were tunneled subcutaneously to the dorsal region of the neck and flushed with 300 ml of saline containing heparin (40 U/ml) and 500 ml of sodium penicillin G (10 000 U/ml). The catheters were then filled with a viscous solution of 50% polyvinylpyrrolidone (PVP) and capped with a piece of polyethylene tubing melted and sealed at one end. After surgery, the rats were kept at the same preoperative conditions. The food intake was recovered 2/ 3 days after surgery. 2.4. Euglycemic clamp procedures One week after surgery, each rat was submitted to a two-step hyperinsulinemic euglycemic clamp procedure, after an overnight fast to assess the whole-body insulin action. The rat was placed in a restraining cage and extension tubing was attached to the jugular catheter by an adapter for the continuous infusion of insulin and glucose. The carotid catheter was used for blood sampling. A primed insulin infusion was delivered at a rate of 3 mU/kg per min (low-dose) for 90 min and then at an increased rate of 30 mU/kg per min (maximal stimulation, high-dose) for an additional 90 min period. Based on the blood glucose concentration measured every 10 min, the blood glucose was kept constant at the basal level with a variable infusion of a 20% (w/v) glucose solution. Additional blood samples were collected just before insulin infusions and at 90 and 180 min after starting insulin infusion for the determination of the plasma insulin concentrations. The glucose infusion rate (GIR) in mg/kg per min was calculated every 10 min during the clamp study period. The means of GIR values from 60 to 90 min and from 150 to 180 min for the two-step sequential euglycemic clamp procedure were regarded as an index of the whole body insulin action since a plateau in the GIR was achieved during these periods of time, as reported previously [15 /17]. At the end of the euglycemic clamp, skeletal muscle was excised, made free of blood, and immediately frozen at liquid nitrogen temperature before it was stored at /80 8C until analysis.
141
2.5. Blood assays Blood glucose concentration was determined using a glucose analyzer (model 2300A; Yellow Springs Instrument, OH). Plasma insulin was assayed with a radioimmunoassay kit (Phadesepa Insulin RIA, Pharmacia AB, Sweden). Plasma free fatty acids (FFA) were determined with a commercial kit (Wako pure Chemical Industries, Osaka, Japan).
2.6. Tissue processing Muscle samples were homogenized in ice-cold homogenizing buffer (20 mM Tris /Cl, pH 7.6, 150 mM NaCl, 1% Nonidet P-40, 10 mM NaF, 10 mM Na3VO4, 10 mM EDTA, 1 mM PMSF, 1 mg/ml Leupeptin, and 1 mg/ml Aprotinin) using a Polytron homogenizer as described by [18]. The homogenates were kept at ice temperature for 1 h and then centrifuged at 38 000 rpm (150 000 /g ) at 4 8C for 1 h using a Hitachi RP55 rotor (Hitachi, Tokyo, Japan), the protein concentration of the supernatants was determined using a commercial kit (Bio-Rad, Richmond, CA). Supernatant was stored at /80 8C until used.
2.7. Protein expression For the assays of the protein expression of the IR-b, IRS-1, and PI3-kinase, an aliquot (40 mg) of the supernatant was resuspended in treatment buffer containing b-mercaptoethanol and boiled for 5 min. The supernatant proteins (40 mg) were size-fractionated by SDS-PAGE (6 or 10% acrylamide gels). After electroblotting, the PVDF membranes were incubated overnight with the anti-IR-b, anti-IRS-1, or anti-PI3-kinase antibodies at 4 8C. The membrane was then incubated with Goat Anti-Rabbit IgG (1:2000) in Tris / buffer (pH 7.4) containing 3% BSA for 1 h at room temperature. After washing, blotted proteins were visualized using a Western blotting detection system (ECL Plus, Amersham, UK). Quantification of the band intensity was performed using the public domain NIH image software.
142
B. Qin et al. / Diabetes Research and Clinical Practice 62 (2003) 139 /148
2.8. Insulin receptor-b and IRS-1 tyrosine phosphorylation and IRS-1 associated with PI 3kinase The supernatants containing equal amounts of protein (1 mg/ml each tube) were incubated overnight with anti-IR-b (5 mg/ml) or anti-IRS-1 (5 mg/ml) at 4 8C, as indicated, and then with 20 ml of protein A agarose beads at 4 8C for 4 h. The immune complexes were washed as described by [11]. Samples were resuspended in treatment buffer containing b-mercaptoethanol and boiled for 5 min. Phosphorylated proteins were separated by SDS-PAGE (6 or 10% acrylamide gels). After electroblotting, the PVDF membranes were incubated with phosphotyrosine antibody or PI3kinase antibody. Phosphorylated proteins were visualized by the same method mentioned above. 2.9. Statistical analysis All data are expressed as mean9/S.E. Data were analyzed by the one-way analysis of variance. When a significant difference was found (P B/ 0.05), the results were further compared with the Fisher’s PLSD test. The StatView 5.0 software (SAS Institute Inc., Cary, NC) was used for the statistical analysis.
3. Results 3.1. Body weight, plasma FFA, blood glucose, and plasma insulin levels The body weight, plasma FFA, blood glucose, and plasma insulin levels before, during and immediately after the euglycemic clamp are shown in Table 1. The final body weights of the cinnamon treated rats and the controls were not significantly different. Although body weights were decreased in all rats after surgery for cannulation, they returned to preoperative levels after 3 days. Cinnamon treatment for 3 weeks did not affect plasma FFA and fasting blood glucose concentrations in either 30 or 300-mg/kg dose treated rats. Basal and steady state plasma insulin concentrations during the low-dose and the high-dose
insulin clamp procedures were not significantly different among all experimental groups; namely, 3-weeks cinnamon extract administration did not affect insulin secretion. 3.2. Effect of cinnamon extract administration on GIRs The average GIRs for the last 30 min during the low-dose and high-dose insulin infusion rates are shown in Figs. 1 and 2. Because a plateau GIR was achieved, GIR was used as an indicator of wholebody glucose utilization. At the low-dose insulin clamp studies, the GIRs of the cinnamon extractadministered groups were significantly increased (30 mg/kg dose: 118% of controls, P B/0.05; 300 mg/kg dose: 146% of controls, P B/0.001), as shown in Fig. 2. In the high-dose insulin infusion, the GIR of the rats treated with a 300-mg/kg dose of cinnamon extract was also significantly greater than that of controls (117% of controls, P B/0.05). However, a 30-mg/kg dose did not affect the GIR at the high-dose clamp. 3.3. Effect of cinnamon extract administration on IR-b, IRS-1, and PI 3-kinase protein contents in rat skeletal muscle The total protein content of IR-b, IRS-1, and PI 3-kinase was measured in skeletal muscles of all rats. No significant difference in the protein content of IR-b, IRS-1, and PI 3-kinase was detected (Fig. 3A, Fig. 4A, Fig. 5A, respectively). 3.4. Effect of cinnamon extract administration on tyrosine-phosphorylation of IR-b and IRS-1, and the IRS-1 association with PI 3-kinase in rat skeletal muscle The tyrosine phosphorylation level of IR-b was determined by immunoblotting the phosphotyrosine antibody immunoprecipitates with the IR-b antibody. As shown in Fig. 3B, the tyrosine phosphorylation level of IR-b in skeletal muscle of the cinnamon extract administered animals was significantly increased when compared with controls (118% of control, P B/0.05). The same tendency was found for the IRS-1 tyrosine phos-
B. Qin et al. / Diabetes Research and Clinical Practice 62 (2003) 139 /148
143
Table 1 Body weight, plasma FFA, and concentrations of blood glucose and plasma insulin before and immediately after the euglycemic clamp procedure at low-dose and high-dose insulin infusion Group
Saline Cinnamon (30 mg/kg BW) Cinnamon (300 mg/kg BW)
Body weight (g)
2719/3 2759/4 2729/5
FFA (mEq/l)
0.649/0.04 0.669/0.05 0.689/0.06
phorylation (133% of controls, P B/0.01), as shown in Fig. 4B. The IRS-1/PI 3-kinase association level was determined by immunoblotting the PI 3-kinase antibody immunoprecipitates with the IRS-1 antibody. As shown in Fig. 5B, the IRS-1/PI 3-kinase association had a significant increase in the skeletal muscles of cinnamon extract treated animals (41% above controls, P B/0.01).
4. Discussion In vitro studies have suggested that the cinnamon extract acts as an insulin mimetic, enhancing the glucose uptake in adipocytes [3 /6]. Addition-
Glucose (mg/dl)
Insulin (mU/ml)
Basal
3.0
30.0
Basal
3.0
30.0
759/2 739/3 719/3
749/3 749/2 759/2
739/1 739/4 729/3
7.89/0.1 8.09/0.3 7.99/0.2
299/3 319/2 309/2
5809/15 6129/19 6059/25
ally, treatment with cinnamon extract has been reported to enhance IR kinase activity, autophosphorylation of IR, glycogen synthesis, and glycogen synthase activity in 3T3-L1 adipocytes [6]. The present study was undertaken to assess the effect of different doses of cinnamon extract on the insulin action in vivo in the conscious unstressed rats. We investigated the insulin-induced glucose uptake using a two-step hyperinsulinemic euglycemic clamp procedure. Researches on the wholebody insulin-mediated glucose utilization under conditions of euglycemic hyperinsulinemia suggested that the net splanchnic glucose uptake is similar in control and type 2 diabetes mellitus subjects; in addition, adipose tissue glucose uptake
Fig. 1. GIRs for the euglycemic clamp procedure period at the low-dose (3.0 mU/kg per min) and the high-dose (30.0 mU/kg per min) insulin infusion for animals treated with saline, 30 and 300-mg/kg cinnamon extract dose. (m) Saline treated; (") 30-mg/kg dose cinnamon extract treated; (k) 300-mg/kg dose cinnamon extract treated. Values are mean9/S.E.
144
B. Qin et al. / Diabetes Research and Clinical Practice 62 (2003) 139 /148
Fig. 2. GIR during the euglycemic clamp procedure in normal control and cinnamon treated (30 and 300-mg/kg BW, respectively), rats at the low-dose (3.0 mU/kg per min) and high-dose (30.0 mU/kg per min) insulin infusion. Data are expressed as the mean9/S.E. for six rats in each group. *, P B/0.05 vs. control; **, P B/0.001 vs. control.
is assumed to represent /2% of the total glucose disposal [19,20]. Muscle glucose uptake in control subjects represents /75% of the total glucose metabolism [13,14,21] and in type 2 diabetic subjects accounts for essentially all of the impairment in insulin-mediated glucose uptake. In other words, the insulin-stimulated glucose disposal during the euglycemic clamp is mainly credited to the glucose uptake in skeletal muscle. During this study, a high physiological insulin level of
about 30 mU/ml achieved during low-dose insulin infusion rate reflects insulin sensitivity in peripheral tissues (i.e. skeletal muscle) and liver in the postprandial state. Changes in GIR are thought to be caused mainly by changes in the insulin receptor binding at high physiological insulin levels. Although we did not measure the effect of insulin on hepatic glucose production (HGP), according to Tominaga et al. [22], HGP can be completely suppressed under an insulin infusion
Fig. 3. Effect of 3-weeks 300-mg/kg BW dose cinnamon extract administration on IR-b protein content (A) and IR-b tyrosine phosphorylation (B) in gastrocnemius of rats. Muscle proteins were resolved by 10% SDS-PAGE, and tyrosine phosphorylation was detected by immunoprecipitation with anti-IR- antibody followed by immunoblot analysis with antiphosphotyrosine antibody. Data are expressed as the mean9/S.E. for five rats in each group. *, P B/0.05 vs. controls.
B. Qin et al. / Diabetes Research and Clinical Practice 62 (2003) 139 /148
145
Fig. 4. Effect of 3-weeks 300-mg/kg BW dose cinnamon extract administration on IRS-1 protein content (A) and IRS-1 tyrosine phosphorylation (B) in gastrocnemius of rats. Muscle proteins were resolved by 6% SDS-PAGE, and tyrosine phosphorylation was detected by immunoprecipitation with anti-IRS-1 antibody followed by immunoblot analysis with antiphosphotyrosine antibody. Data are expressed as the mean9/S.E. for five rats in each group. *, P B/0.01 vs. controls.
Fig. 5. Effect of 3-weeks 300-mg/kg BW dose cinnamon extract administration on PI 3-kinase protein content (A) and IRS-1/PI 3kinase association (B) in gastrocnemius of rats. Muscle proteins were resolved by 10% SDS-PAGE, and IRS-1/PI 3-kinase association was detected by immunoprecipitation with anti-IRS-1 antibody followed by immunoblot analysis with anti-PI 3-kinase antibody. Data are expressed as the mean9/S.E. for five rats in each group. *, P B/0.01 vs. controls.
146
B. Qin et al. / Diabetes Research and Clinical Practice 62 (2003) 139 /148
rate of 3 mU/kg per min, and an even lower infusion rate (2.4 mU/kg per min) was shown to suppress around 90% of the HGP in conscious unstressed rats [23], therefore, the GIR is essentially synonymous with the rate of the total body glucose utilization [23]. Naturally, these effects of insulin infusion on HGP or its action on peripheral tissues are passive of change according to the experimental conditions (i.e. type of animal, sex, age, diet, anesthesia or awake condition, and stress), for example, Lam et al. [24] reported that 5 mU/kg per min of insulin infusion suppressed endogenous glucose production (EGP) by /55% only. EGP was inhibited by insulin in a dosedependent fashion [23], the high-dose insulin infusion leads to a maximal insulin action and insulin responsiveness, predominantly indicating the capacity of post-receptor binding mechanisms [25,26], and resulted in the pharmacological insulin range, which sufficiently suppresses HGP [23]. Therefore, the results of the present study suggest that cinnamon extract administration would improve the insulin sensitivity and responsiveness in peripheral tissues in a dose-dependent manner (Figs. 1 and 2). The insulin signaling pathway leading to the cellular uptake of glucose begins with the binding of insulin to its receptor (IR); in the sequence, ligand binding to the IR induces autophosphorylation of specific tyrosine residues, which then activates the substrate protein tyrosine kinase of the IR [27]. The activated IR phosphorylates several intracellular proteins, including the IRS1. Tyrosine phosphorylation of the IRS-1 leads to the binding of PI 3-kinase [28] and the activation of its enzymatic activity, a necessary step for the translocation of glucose transporter-4 to the plasma membrane, consequently elevating the rate of cellular glucose uptake in response to insulin [29]. Reduced IRS-1 expression was observed in Zucker fatty rats [30], and mice that are deficient in the IRS-1, prepared by target geneknockout, exhibited hyperinsulinemia and glucose intolerance [31 /33]. These findings propose that decreased IRS-1 protein expression is involved in the development of insulin resistance. On the other hand, several defects in insulin signaling have been reported in type 2 diabetes, including a modest
reduction in insulin receptor phosphorylation and tyrosine kinase activity [34], decrease in insulinstimulated IRS-1 tyrosine phosphorylation [35,36], and reduced PI 3-kinase activity [35 /37]. However, the IRS-1 protein expression appears to be unchanged in these studies [35 /37]. Regardless of differences between humans and animals or the progress of specific diseases, the decrease in insulin effect is unquestionably caused by changes in insulin signaling (protein expression and/or phosphorylation level). Therefore, we further clarified the effect of cinnamon extract treatment on the molecular mechanisms of the in vivo insulin signaling in skeletal muscle obtained from the experimental rats. Our data show that cinnamon extract treatment does not affect the IRS-1 protein amount (Fig. 4A), but significantly enhances the tyrosine phosphorylation level of IRS-1 (Fig. 4B). Additionally, the same trend was found in the IRb, the metabolic upstream effects of IRS-1 (Fig. 3B), in good agreement with the result reported by Imparl-Radosevich et al. [5]. The activation of PI 3-kinase is required for insulin and stimulates the glucose transport [38,39]. Although we have not assessed directly the effect on the activation of PI 3-kinase, it was previously described that, in parallel with an increase in IRS-1/PI 3-kinase association in skeletal muscle of STZ-diabetes [40], there is an increase in insulin-stimulated IRS-1 tyrosine phosphorylation and IRS-1-associated PI 3-kinase activity [40]. To be precise, PI 3-kinase activation closely correlates with IRS-1 phosphorylation [40]. The present data revealed that cinnamon extract treatment increases IRS-1 tyrosine phosphorylation levels and the IRS-1/PI 3-kinase association (Fig. 5B); therefore, these changes are thought to result in improved activation of PI 3-kinase. In summary, the present study showed for the first time that (1) oral treatment with the cinnamon extract enhances the glucose utilization in vivo, and this effect occurs in a dose-dependent manner; (2) the cinnamon extract potentiates the insulin-stimulated tyrosine phosphorylation of IRb and IRS-1 and the IRS-1 association with PI 3kinase, however, without affecting their protein content levels.
B. Qin et al. / Diabetes Research and Clinical Practice 62 (2003) 139 /148
In conclusion, the current study suggests that the oral treatment with cinnamon extract would improve in vivo insulin-regulated whole-body glucose utilization in a dose-dependent fashion in rats, at least in part through enhancing the insulin signaling in skeletal muscle. Further investigation on the effect of the cinnamon extract on the adultonset of diabetes is needed.
Acknowledgements This work was supported in part by Grants of the Research from Projects on Aging and Health from the Ministry of Health, Welfare and Labor of Japan (H13-009) and from the Kampo Science Foundation of Japan (2002).
References [1] F.S. Leung, A.Y., in: Encyclopedia of common natural ingredients used in foods, drugs, and cosmetics, second ed., Wiley, New York, 1996, pp. 168 /170. [2] K. Toriizuka, Basic lecture of Kampo medicine: pharmacological effect of cinnamon, Kampo Med. 11 (1998) 431 / 436. [3] C.L. Broadhurst, M.M. Polansky, R.A. Anderson, Insulinlike biological activity of culinary and medicinal plant aqueous extracts in vitro, J. Agric. Food Chem. 48 (2000) 849 /852. [4] A. Khan, N.A. Bryden, M.M. Polansky, R.A. Anderson, Insulin potentiating factor and chromium content of selected foods and spices, Biol. Trace Elem. Res. 24 (1990) 183 /188. [5] J. Imparl-Radosevich, S. Deas, M.M. Polansky, D.A. Baedke, T.S. Ingebritsen, R.A. Anderson, D.J. Graves, Regulation of PTP-1 and insulin receptor kinase by fractions from cinnamon: implications for cinnamon regulation of insulin signalling, Horm. Res. 50 (1998) 177 /182. [6] K.J. Jarvill-Taylor, R.A. Anderson, D.J. Graves, A hydroxychalcone derived from cinnamon functions as a mimetic for insulin in 3T3-L1 adipocytes, J. Am. Coll. Nutr. 20 (2001) 327 /336. [7] M.P. Czech, Molecular actions of insulin on glucose transport, Annu. Rev. Nutr. 15 (1995) 441 /471. [8] G.M. Reaven, Pathophysiology of insulin resistance in human disease, Physiol. Rev. 75 (1995) 473 /486. [9] C.R. Kahn, Banting lecture. Insulin action, diabetogenes, and the cause of type 2 diabetes, Diabetes 43 (1994) 1066 / 1084.
147
[10] J.M. Olefsky, O.G. Kolterman, J.A. Scarlett, Insulin action and resistance in obesity and noninsulin-dependent type 2 diabetes mellitus, Am. J. Physiol. 243 (1982) E15 / E30. [11] L.J. Goodyear, F. Giorgino, L.A. Sherman, J. Carey, R.J. Smith, G.L. Dohm, Insulin receptor phosphorylation, insulin receptor substrate-1 phosphorylation, and phosphatidylinositol 3-kinase activity are decreased in intact skeletal muscle strips from obese subjects, J. Clin. Invest. 95 (1995) 2195 /2204. [12] M. Bjornholm, Y. Kawano, M. Lehtihet, J.R. Zierath, Insulin receptor substrate-1 phosphorylation and phosphatidylinositol 3-kinase activity in skeletal muscle from NIDDM subjects after in vivo insulin stimulation, Diabetes 46 (1997) 524 /527. [13] R.A. DeFronzo, E. Jacot, E. Jequier, E. Maeder, J. Wahren, J.P. Felber, The effect of insulin on the disposal of intravenous glucose. Results from indirect calorimetry and hepatic and femoral venous catheterization, Diabetes 30 (1981) 1000 /1007. [14] R.A. DeFronzo, R. Gunnarsson, O. Bjorkman, M. Olsson, J. Wahren, Effects of insulin on peripheral and splanchnic glucose metabolism in noninsulin-dependent (type 2) diabetes mellitus, J. Clin. Invest. 76 (1985) 149 /155. [15] L. Li, Y. Oshida, M. Kusunoki, K. Yamanouchi, B.L. Johansson, J. Wahren, Y. Sato, Rat C peptide I and II stimulate glucose utilization in STZ-induced diabetic rats, Diabetologia 42 (1999) 958 /964. [16] Y. Oshida, Y. Tachi, Y. Morishita, K. Kitakoshi, N. Fuku, Y.Q. Han, I. Ohsawa, Y. Sato, Nitric oxide decreases insulin resistance induced by high-fructose feeding, Horm. Metab. Res. 32 (2000) 339 /342. [17] X. Hu, J. Sato, Y. Oshida, M. Xu, G. Bajotto, Y. Sato, Effect of Gosha-jinki-gan (chinese herbal medicine: NiuChe-Sen-Qi-Wan) on insulin resistance in streptozotocininduced diabetic rats, Diabetes Res. Clin. Pract. 59 (2003) 103 /111. [18] M. Nagasaki, N. Nakai, Y. Oshida, Z. Li, M. Xu, M. Obayashi, T. Murakami, A. Yoshimura, N. Fujitsuka, Y. Shimomura, Y. Sato, Exercise training prevents maturation-induced decreases in insulin receptor substrate-1 and phosphatidylinositol 3-kinase in rat skeletal muscle, Metabolism 49 (2000) 954 /959. [19] P. Bjorntorp, L. Sjostrom, Carbohydrate storage in man: speculations and some quantitative considerations, Metabolism 27 (1978) 1853 /1865. [20] P. Marin, M. Rebuffe-Scrive, U. Smith, P. Bjorntorp, Glucose uptake in human adipose tissue, Metabolism 36 (1987) 1154 /1160. [21] R.A. DeFronzo, Lilly lecture, The triumvirate: beta-cell, muscle, liver. A collusion responsible for NIDDM, Diabetes 37 (1988) (1987) 667 /687. [22] M. Tominaga, M. Kimura, K. Sugiyama, T. Abe, K. Igarashi, M. Igarashi, H. Eguchi, A. Sekikawa, A. Ogawa, H. Manaka, et al., Effects of seishin-renshi-in and Gymnema sylvestre on insulin resistance in streptozotocin-
148
[23]
[24]
[25]
[26]
[27]
[28]
[29]
[30]
[31]
[32]
B. Qin et al. / Diabetes Research and Clinical Practice 62 (2003) 139 /148 induced diabetic rats, Diabetes Res. Clin. Pract. 29 (1993) 11 /17. D. Smith, L. Rossetti, E. Ferrannini, C.M. Johnson, C. Cobelli, G. Toffolo, L.D. Katz, R.A. DeFronzo, In vivo glucose metabolism in the awake rat: tracer and insulin clamp studies, Metabolism 36 (1987) 1167 /1174. T.K. Lam, H. Yoshii, C.A. Haber, E. Bogdanovic, L. Lam, I.G. Fantus, A. Giacca, Free fatty acid-induced hepatic insulin resistance: a potential role for protein kinase C-delta, Am. J. Physiol. Endocrinol. Metab. 283 (2002) E682 /E691. C.R. Khan, Insulin resistance, insulin insensitivity and insulin unresponsiveness: a necessary distinction, Metabolism 27 (1978) 1893 /1902. J.M. Olefsky, O.G. Kolterman, J.A. Scarlett, Insulin action and resistance in obesity and noninsulin-dependent type 2 diabetes mellitus, Am. J. Physiol. 243 (1999) E15 / E30. C.R. Kahn, M.F. White, The insulin receptor and the molecular mechanism of insulin action, J. Clin. Invest. 82 (1988) 1151 /1156. M.F. White, The IRS-signalling system: a network of docking proteins that mediate insulin action, Mol. Cell. Biochem. 182 (1998) 3 /11. K. Hara, K. Yonezawa, H. Sakaue, A. Ando, K. Kotani, T. Kitamura, Y. Kitamura, H. Ueda, L. Stephens, T.R. Jackson, et al., 1-Phosphatidylinositol 3-kinase activity is required for insulin-stimulated glucose transport but not for RAS activation in CHO cells, Proc. Natl. Acad. Sci. USA 91 (1994) 7415 /7419. M. Anai, M. Funaki, T. Ogihara, J. Terasaki, K. Inukai, H. Katagiri, Y. Fukushima, Y. Yazaki, M. Kikuchi, Y. Oka, T. Asano, Altered expression levels and impaired steps in the pathway to phosphatidylinositol 3-kinase activation via insulin receptor substrates 1 and 2 in Zucker fatty rats, Diabetes 47 (1998) 13 /23. E. Araki, M.A. Lipes, M.E. Patti, J.C. Bruning, B.R. Haag, R.S. Johnson, C.R. Kahn, Alternative pathway of insulin signalling in mice with targeted disruption of the IRS-1 gene, Nature 372 (1994) 186 /190. H. Tamemoto, T. Kadowaki, K. Tobe, T. Yagi, H. Sakura, T. Hayakawa, Y. Terauchi, K. Ueki, Y. Kaburagi, S. Satoh, et al., Insulin resistance and growth retardation in mice lacking insulin receptor substrate-1, Nature 372 (1994) 182 /186.
[33] T. Yamauchi, K. Tobe, H. Tamemoto, K. Ueki, Y. Kaburagi, R. Yamamoto-Honda, Y. Takahashi, F. Yoshizawa, S. Aizawa, Y. Akanuma, N. Sonenberg, Y. Yazaki, T. Kadowaki, Insulin signalling and insulin actions in the muscles and livers of insulin-resistant, insulin receptor substrate 1-deficient mice, Mol. Cell. Biol. 16 (1996) 3074 / 3084. [34] A. Handberg, A. Vaag, J. Vinten, H. Beck-Nielsen, Decreased tyrosine kinase activity in partially purified insulin receptors from muscle of young, non-obese first degree relatives of patients with type 2 (non-insulindependent) diabetes mellitus, Diabetologia 36 (1993) 668 /674. [35] W. Pratipanawatr, T. Pratipanawatr, K. Cusi, R. Berria, J.M. Adams, C.P. Jenkinson, K. Maezono, R.A. DeFronzo, L.J. Mandarino, Skeletal muscle insulin resistance in normoglycemic subjects with a strong family history of type 2 diabetes is associated with decreased insulinstimulated insulin receptor substrate-1 tyrosine phosphorylation, Diabetes 50 (2001) 2572 /2578. [36] H. Storgaard, X.M. Song, C.B. Jensen, S. Madsbad, M. Bjornholm, A. Vaag, J.R. Zierath, Insulin signal transduction in skeletal muscle from glucose-intolerant relatives of type 2 diabetic patients [corrected], Diabetes 50 (2001) 2770 /2778. [37] K. Cusi, K. Maezono, A. Osman, M. Pendergrass, M.E. Patti, T. Pratipanawatr, R.A. DeFronzo, C.R. Kahn, L.J. Mandarino, Insulin resistance differentially affects the PI 3-kinase- and MAP kinase-mediated signaling in human muscle, J. Clin. Invest. 105 (2000) 311 /320. [38] B. Cheatham, C.J. Vlahos, L. Cheatham, L. Wang, J. Blenis, C.R. Kahn, Phosphatidylinositol 3-kinase activation is required for insulin stimulation of pp70 S6 kinase, DNA synthesis, and glucose transporter translocation, Mol. Cell. Biol. 14 (1994) 4902 /4911. [39] T. Okada, Y. Kawano, T. Sakakibara, O. Hazeki, M. Ui, Essential role of phosphatidylinositol 3-kinase in insulininduced glucose transport and antilipolysis in rat adipocytes. Studies with a selective inhibitor wortmannin, J. Biol. Chem. 269 (1994) 3568 /3573. [40] F. Folli, M.J. Saad, J.M. Backer, C.R. Kahn, Regulation of phosphatidylinositol 3-kinase activity in liver and muscle of animal models of insulin-resistant and insulindeficient diabetes mellitus, J. Clin. Invest. 92 (1993) 1787 / 1794.