CHINESE JOURNAL OF COMPARATIVE MEDICINE May, 2019 Vol. 29 No. 5,. 2 PI3K / Akt [J]., 2019, 29 (5): Chen J, Liu YR. Effect of curcum

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1 CHINESE JOURNAL OF COMPARATIVE MEDICINE May, 2019 Vol. 29 No. 5,. 2 PI3K / Akt [J]., 2019, 29 (5): Chen J, Liu YR. Effect of curcumin on glucose transport and PI3K / Akt signaling pathway in adipocytes of type 2 diabetes mellitus rats [J]. Chin J Comp Med, 2019, 29(5): doi: / j.issn PI3K / Akt 1, 2 (1.,430074; 2.,430061) 2 (T2DM) -3( PI3K) / B(Akt) 90 SD ( ) (50 mg / kg) (150 mg / kg) (250 mg / kg) (1. 35 mg / kg),15 2 8,8 12 h, (FBG) ( FINS) ( HOMA-IR),( TG) / (LDC-C / HDL-C) (TC), 4(GluT4), PCR(qRT-PCR)GluT4, GluT4 p-pi3k 2(Irs2) p-akt, 2,8 ;, 2 8 (P<0. 05), FBG FINS HOMA-IR HDC-L LDC-L TC TG, ; GluT4 mrna,glut4 Irs2 p-pi3k / PI3K pakt / Akt ; (P<0. 05), FBG FINS HOMA-IR HDC-L LDC-L TC TG ; ;GluT4 mrna,glut4 Irs2 p-pi3k / PI3K pakt / Akt ; (P<0. 05),(P<0. 05) T2DM, PI3K / Akt GluT4,, / B ; ;2 ;; ; R-33 A (2019) Effect of curcumin on glucose transport and PI3K / Akt signaling pathway in adipocytes of type 2 diabetes mellitus rats CHEN Jie 1, LIU Yiran 2 (1. School of Biological Engineering, Wuhan Polytechnic, Wuhan , China. 2. Department of Acupuncture, Hospital of Traditional Chinese Medical in Hubei, Wuhan ) [] (B ):2 [](1980 ),,,,: chenjie9180@ 163.com

2 91 Abstract Objective To investigate the effects of curcumin on glucose transport and phosphatidylinositol 3- kinase (PI3K) / protein kinase B (Akt) signaling pathway in adipocytes of type 2 diabetes mellitus (T2DM) rats. Methods Ninety Sprague-Dawley ( SD) rats were randomly divided into normal group, model group ( high-fat diet-fed), curcumin low-dose group (50 mg / kg), curcumin medium-dose group (150 mg / kg), and curcumin high-dose group (250 mg / kg) groups, and rosiglitazone group ( mg / kg), each group comprised 15 rats. The body weight of rats was measured immediately after administration, 2 weeks after administration and 8 weeks after administration, and blood was collected for 12 hours after fasting at the end of 8 weeks, fasting blood glucose (FBG), fasting insulin (FINS), the insulin resistance index ( HOMA-IR), triglyceride ( TG), low / high-density lipoprotein cholesterol ( LDC-C / HDL-C), and total cholesterol ( TC) were measured. The glucose perfusion rate was measured by positive glucose clamp assay, the translocation of glucose transporter 4 ( GluT4) in adipocyte membrane was detected by fluorescence immunoassay,glut4 in adipocytes was detected by real-time fluorescence quantitative PCR ( qrt-pcr), the expression of GluT4, p-pi3k, insulin receptor substrate 2 (Irs2) and p-akt in intracellular and extracellular membranes of adipocytes were detected by Western blotting. Results Compared with the normal group, the weight of the model group increased immediately and 2 weeks after administration, and decreased 8 weeks after administration; compared with the model group, the body weight of curcumin treatment group and rosiglitazone treatment group decreased immediately, 2 weeks and 8 weeks after administration ( P < 0. 05). Compared with the normal group, the FBG, FINS, HOMA-IR, HDC-L, LDC-L, TC and TG in the model group increased, and the glucose injection rate decreased in a dose-dependent manner; GluT4 mrna increased in the model group, the protein expression of GluT4, Irs2, p-pi3k / PI3K and pakt / Akt decreased significantly; and the differences were statistically significant (P < 0. 05). Compared with the model group, the levels of FBG, FINS, HOMA-IR, HDC-L, LDC-L, TC and TG in the curcumin treatment group and rosiglitazone group were significantly lower, the rate of glucose injection was significantly higher, GluT4 mrna decreased, the protein expression of GluT4, Irs2, p-pi3k / PI3K and pakt / Akt increased significantly, and the differences were statistically significant ( P< 0. 05), and the difference of curcumin dosage among the three groups was also statistically significant ( P < 0. 05). Conclusions Curcumin can increase the insulin sensitivity of T2DM rats, which may be achieved by activating PI3K / Akt signaling pathway to promote the GluT4 membrane translocation in adipocytes and increase the uptake of glucose by adipocytes, thereby alleviating the balance of blood sugar metabolism. Keywords phosphatidylinositol 3-kinase / protein kinase B signaling pathway; curcumin; type 2 diabetes mellitus; adipocyte; glucose transport; rat 2 ( type 2 diabetes mellitus,t2dm), 90%, [1] - 3( phosphatidylinositol 3-kinase, PI3K) / B (protein kinase B,Akt), T2DM [2] [3],, [4] PI3K / Akt, T2DM,, PI3K / Akt SPF SD 90,75 ~ 100 g,4, [SCXK( ) ], [ SYXK( ) ], 25,50%,, (IACUC :IACUC ) 1. 2 Sigma ; (,:D ); - Amresco ; 4 ( glucose transporter 4, GLUT4) β-actin GluT4 Abcam ;p-pi3k 2(insulin receptor substrate-2,irs2) p-akt SantaCruz ; IgG ;BCA ;RNA ;

3 92 Genecopoeia ;CFX96 PCR Bio-Rad ;CX23 ;GIS-500 Miulab T2DM 75, (66. 5% 2. 5%, 1%,30%),, 12 h,30 mg / kg,72 h, (Fasting blood glucose,fbg), FBG mmol / L [5] 15, -,3R , , [6] 6,15 : ; mg / kg; mg / kg 1, ( 8 ) 12 h,1 ml, 3000 rpm 10 min, -20, FBG (fasting insulin, FINS ), ( homeostasis model assessment for insulin resistance, HOMA-IR ); (triglyceride,tg) / ( low / high density lipoprotein cholesterol, LDC-C / HDL-C) (total cholesterol,tc) Primer names Direction , 5 Keaegen [7] 10% 24 mu / kg / min, 10,, 1 h, ,,4% GluT4 5,, -80,, GluT Real-time PCR GluT4 mrna , 40 ml TES, 4,, TRIzol RNA,RNA RNA cdna, GADPH,2 -ΔΔCq mrna RT-PCR Western blot GluT4 p-pi3k Irs2 p-akt , 40 ml TES, 4,,,1 ml TES r / min 15 min,75 min,,1 ml TES, Western blot GluT4 p-pi3k PI3K Irs2 p-akt Akt,Image-J 1 Real-time PCR Table 1 Real-time PCR primer sequences (5 3 ) Sequences(5 3 ) GluT4 F CACTGGTCCTTGCTGTATTC R CTGATGTTAGCCCTGAGTAC GADPH F TGTGTCCGTCGTGGATCTGA R TTGCTGTTGAAGTCGCAGGAG

4 SPSS21. 0, ± ( x ± s ),,q,p< ,,,;,,,;,,, 2,8 ;, 2 8 (P<0. 05) FBG FINS HOMA-IR Groups Table 2, FBG FINS HOMA-IR ;, FBG FINS HOMA-IR,( P< 0. 05) , HDC-L LDC-L TC TG ;, HDC-L LDC-L TC TG (P<0. 05) ,;,, ( P < 0. 05) GluT4, GluT4, GluT4, 1 2 ( x ± s, n = 15,g) Body weight changes in the rats of each experimental group after modeling Immediate medication 2 Medication for 2 weeks 8 Medication for 8 weeks Normal group ± ± ± Model group ± ± ± Low dose curcumin group Medium dose curcumin group High dose curcumin group Rosiglitazone group ± # ± # ± # ± # ± # ± # ± # ± # ± # ± # ± # ± # :, P<0. 05;, # P<0. 05 Note. Compared with the normal group, P< Compared with the model group, # P< Table 3 3 FBG FINS HOMA-IR ( x ± s, n = 15) Changes in FBG, FINS and HOMA-IR in the rats of each group after modeling Groups FBG(mmol / L) FINS(mIU / L) HOMA-IR Normal group 5. 81± ± ±0. 07 Model group ± ± ±0. 18 Low dose curcumin group Medium dose curcumin group High dose curcumin group Rosiglitazone group 9. 13±1. 55 # 2. 53±0. 47 # 0. 98±0. 17 # 8. 06±1. 47 # 2. 15±0. 48 #a 0. 84±0. 15 #a 6. 51±1. 16 #ab 1. 44±0. 39 #ab 0. 68±0. 13 #ab 5. 62±0. 89 # 1. 27±0. 35 # 0. 61±0. 12 # :, P<0. 05;, # P<0. 05;, a P<0. 05;, b P<0. 05 Note. Compared with the normal group, P< Compared with the model group, # P< Compared with the low dose curcumin group, a P< Compared with the medium dose curcumin group, b P<0. 05.

5 94 中国比较医学杂志 2019 年 5 月第 29 卷第 5 期组别 Groups Chin J Comp Med, May 2019,Vol. 29,No. 5 表 4 各组大鼠血脂水平测定结果比较( x ± s, n = 15,mmol / L) Table 4 Comparison of blood lipid levels in the rats of each experimental group HDC-L LDC-L TC 正常组 Normal group 0. 91± ± ±0. 16 姜黄素低剂量组 Low dose curcumin group 1. 34±0. 25 # 2. 96±0. 52 # 5. 72±0. 49 # 模型组 Model group 1. 56±0. 28 姜黄素中剂量组 Medium dose curcumin group 姜黄素高剂量组 High dose curcumin group 罗格列酮组 Rosiglitazone group 3. 85±0. 44 TG 0. 69± ± ± ±0. 21 # 1. 16±0. 21 #a 2. 44±0. 47 #a 5. 18±0. 51 #a 0. 96±0. 23 #a 1. 01±0. 18 #ab 1. 86±0. 49 #ab 4. 31±0. 38 #ab 0. 80±0. 16 #ab 0. 96±0. 20 # 1. 62±0. 37 # 3. 75±0. 44 # 0. 73±0. 16 # 注:与正常组相比, P<0. 05;与模型组相比, # P<0. 05;与姜黄素低剂量组相比, a P<0. 05;与姜黄素中剂量组相比, b P<0. 05 Note. Compared with the normal group, P< Compared with the model group, # P< Compared with the low dose curcumin group, a P < Compared with the medium dose curcumin group, b P< 表 5 各组葡萄糖注射速率对比( x ± s, n = 5,mg / (kg min)) Table 5 Comparison of glucose infusion rates in the rats of each group 葡糖糖注射速率组别 Groups Glucose infusion rate 正常组 Normal group ±2. 95 模型组 Model group ±2. 18 姜黄素低剂量组 Low dose curcumin group ±2. 29# 姜黄素中剂量组 Medium dose curcumin group ±2. 66# 姜黄素高剂量组 High dose curcumin group ±2. 45# 罗格列酮组 Rosiglitazone group ±2. 38# 注:与正常组相比, P<0. 05;与模型组相比, # P < 0. 05;与姜黄素低剂量组相比, a P<0. 05;与姜黄素中剂量组相比, b P<0. 05 Note. Compared with the normal group, P < Compared with the model group, # P< Compared with the low dose curcumin group, a P< Compared with the medium dose curcumin group, b P< 脂肪细胞 GluT4 mrna 表达与正常组相比,模型组细胞 GluT4 mrna 表达降低;与模型组相比,姜黄素治疗组罗格列酮组细胞 GluT4 mrna 表达升高, 具有剂量依赖性 ( P < 0. 05) 见表脂肪细胞内外膜 GluT4 Irs2 p-pi3k / PI3K pakt / Akt 表达与正常组相比, 模型组细胞内外膜 GluT4 Irs2 p-pi3k / PI3K pakt / Akt 蛋白表达降低;与模型组相比, 姜黄素治疗组罗格列酮组细胞内外注:A:正常组;B:模型组;C:姜黄素低剂量组;D:姜黄素中剂量组;E:姜黄素高剂量;F:罗格列酮组图1 脂肪细胞 GluT4 免疫荧光检测结果( 1000) Note. A, Normal group; B, Model group; C, Low dose curcumin group; D, Medium dose curcumin group; E, High dose curcumin group; F, Rosiglitazone group. Figure 1 Immunofluorescence analysis of GluT4 localization in adipocytes

95 GluT4 Irs2 p-pi3k / PI3K pakt / Akt,( P < 0. 05) 2 3, 7 8 6 GluT4 mrna ( x ± s, n = 5) Table 6 GluT4 mrna expression in the adipocytes Groups GluT4 mrna Normal group 1. 03±0.

6 95 GluT4 Irs2 p-pi3k / PI3K pakt / Akt,( P < 0. 05) 2 3, GluT4 mrna ( x ± s, n = 5) Table 6 GluT4 mrna expression in the adipocytes Groups GluT4 mrna Normal group 1. 03±0. 08 Model group Low dose curcumin group Medium dose curcumin group 0. 41± ± ±0. 07 #a High dose curcumin group 0. 75±0. 09 #ab Rosiglitazone group 0. 87±0. 11 #ab :, P<0. 05;, # P< 0. 05;, a P<0. 05;, b P<0. 05 Note. Compared with the normal group, P < Compared with the model group, # P < Compared with the low dose curcumin group, a P < Compared with the medium dose curcumin group, b P< :A: ;B: ;C:;D:;E: ;F: 2 GluT4 Irs2 p-pi3k/ PI3K pakt / Akt Note. A, Normal group; B, Model group; C, Low dose curcumin group; D, Medium dose curcumin group; E, High dose curcumin group; F, Rosiglitazone group. Figure 2 GluT4, Irs2, p-pi3k and p-akt protein expressions on the outer membrane of adipocytes 7 GluT4 Irs2 p-pi3k / PI3K pakt / Akt ( x ± s, n = 5) Table 7 GluT4, Irs2, p-pi3k and p-akt protein expressions on the outer membrane of adipocytes Groups GluT4 Irs2 p-pi3k / PI3K p-akt / Akt Normal group 0. 82± ± ± ±0. 15 Model group 0. 14± ± ± ±0. 11 Low dose curcumin group Medium dose curcumin group High dose curcumin group Rosiglitazone group 0. 23±0. 04 # 0. 35±0. 05 # 0. 78± ± ±0. 10 #a 0. 56±0. 11 #a 0. 81±0. 07 # 0. 76±0. 08 # 0. 63±0. 06 #ab 0. 81±0. 09 #ab 0. 85±0. 07 # 0. 78±0. 06 #a 0. 71±0. 04 # 0. 90±0. 11 # 0. 88±0. 07 # 0. 84±0. 07 # :, P<0. 05;, # P<0. 05;, a P<0. 05;, b P<0. 05 Note. Compared with the normal group, P< Compared with the model group, # P< Compared with the low dose curcumin group, a P< Compared with the medium dose curcumin group, b P< GluT4 Irs2 p-pi3k / PI3K pakt / Akt ( x ± s, n = 5) Table 8 GluT4, Irs2, p-pi3k and p-akt protein expressions on the inner membrane of adipocytes Groups GluT4 Irs2 p-pi3k / PIK3 p-akt / Akt Normal group 1. 22± ± ± ±0. 12 Model group 0. 23± ± ± ±0. 08 Low dose curcumin group 0. 46±0. 21 # 0. 38±0. 04 # 0. 65±0. 04 # 0. 75±0. 06 # Medium dose curcumin group 0. 59±0. 12 #a 0. 55±0. 08 #a 0. 78±0. 05 #a 0. 83±0. 07 # High dose curcumin group 0. 91±0. 09 #ab 0. 97±0. 09 #ab 0. 79±0. 06 #a 0. 95±0. 09 #ab Rosiglitazone group 1. 03±0. 13 # 1. 16±0. 11 # 0. 81±0. 05 # 0. 98±0. 13 # :, P<0. 05;, # P<0. 05;, a P<0. 05;, b P<0. 05 Note. Compared with the normal group, P< Compared with the model group, # P< Compared with the low dose curcumin group, a P< Compared with the medium dose curcumin group, b P<0. 05.

7 96 :A: ;B: ;C:;D: ;E: ;F: 3 GluT4 Irs2 p-pi3k / PI3K pakt / Akt Note. A, Normal group; B, Model group; C, Low dose curcumin group; D, Medium dose curcumin group; E, High dose curcumin group; F, Rosiglitazone group. Figure 3 GluT4, Irs2, p-pi3k and p-akt protein expressions on the inner membrane of adipocytes 3,2 8,,T2DM, FBG FINS HOMA-IR HDC-L LDC-L TC TG,,,, T2DM, [8-9],, T2DM,,, [10],, ;,,,,, Panahi [11] [12] GluT4, GluT4,, GlT4, [13], GluT4, GluT4, GluT4, Western blot GluT4, GluT4, GluT4,, IS PI3K PI3K / Akt,, IRS,PI3K,PIK3, Akt,Akt,, [14] Western blot,, Irs2 p-pi3k / PI3K pakt / Akt ; Irs2 p-pi3k / PI3K pakt / Akt,PI3K / Akt, IR,T2DM,, PI3K / Akt,T2DM,,, GluT4,PI3K / Akt, T2DM : [ 1 ],,,. 2 [J]., 2014, 49(5): [ 2 ],,. PI3K / Akt 2 [ J]., 2017,39( 1): [ 3 ],,, T3-L1 [ J].

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