A PRELIMINARY INVESTIGATION OF THE jpp krakow pl

A Preliminary Investigation Of The Jpp Krakow Pl-Free PDF

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unclear however multiple potential mechanisms have been There were 3 43 kcal g in the regular chow diet total energy. described For instance berberine has been reported to improve carbohydrates 68 fats 18 and protein 14 The high fat. the sensitivity of skeletal muscle and liver cells to insulin by diet was a modification of a previously described diet 26 The. increasing IR expression 20 21 Further there is also evidence diet contained 10 egg yolk powder comprising 30 protein. to suggest that berberine may alter the makeup of gut microbia 60 fat and 10 carbohydrate Bozhou Hongri Egg Products. and consequently help alleviate insulin resistance by decreasing Anhui China 20 pure lard Zhumadian Dingsheng Food Co. the exogenous antigens and increasing intestinal short chain Ltd Henan China 10 sugar and 60 regular chow There. fatty acid concentrations 22 We have also recently reported were 4 96 kcal g in the high fat feed total energy carbohydrates. that berberine can reduce palmitate associated lipotoxicity in 44 fats 48 and proteins 8. pancreatic cells in vitro 23 This finding is relevant in the. context of type 2 diabetes and insulin resistance because Oral glucose tolerance testing and sample preparation. lipotoxicity mediated by increased FFA levels is an important. cause of insulin resistance Further research is needed to After 21weeks 5 rats were randomly selected from each. determine the extent to which berberine affects mediators of group and fasted for 8 hours Fasting blood glucose FBG and. insulin signaling in pancreatic islet and cells and thereby 2 h postprandial blood glucose 2hPG after gavage with a 20. better understand the mechanism of action of berberine glucose solution 2 g kg concentrations were measured in tail. The aim of this preliminary study was to examine the effects vein blood samples using a blood glucose meter Abbott. of berberine on mediators of insulin signaling in pancreatic islet Laboratories Chicago IL. and cells and hepatocytes isolated from rats fed a high fat After 22 weeks all rats were fasted for 8 hours and. diet known to induce obesity and insulin resistance We anesthetized by intraperitoneal injection of 10 chloral hydrate. hypothesized that berberine would ameliorate the development 2 mL kg Thoracotomy was performed and blood was sampled. of insulin resistance and that this may be reflected by differential from the heart An aliquot of blood was used for immediate. expression of insulin IR and IRS 1 in pancreatic cells assessment of FBG concentrations while serum was separated. glucagon IR IRS 1 and IRS 2 in pancreatic cells and IR from the remaining blood and stored at 60 C for later. and IRS 1 in hepatocytes We also examined key metabolic and measurement of fasting insulin FINS fasting glucagon. biochemical indicators of insulin glucose and FFA homeostasis FGLC triglyceride TG and FFA concentrations. The pancreas and liver were then immediately dissected. fixed in 4 formaldehyde and embedded in paraffin for. MATERIALS AND METHODS immunohistochemical detection of pancreatic insulin IR and. IRS 1 and hepatic IR and IRS 1, Animals Visceral fat VF including the epididymal fat pad. retroperitoneal perirenal fat and mesenteric fat was dissected. A total of 49 eight week old male Wistar rats SPF grade and dried on filter paper The weight was recorded and the ratio. weight 160 to 180 g were purchased from Chengdu ASUS of VF to BW VF BW was calculated The insulin resistance. Animal Ltd Sichuan China and housed temperature 18 to index HOMA IR FINS FBG 22 5 was calculated to evaluate. 25 C 12 hour light dark cycle for 2 weeks at the SPF animal the degree of peripheral insulin resistance. facility of West China Hospital of Sichuan University This study. was approved by the Animal Ethics Committee of Sichuan Assays. University, 1 Serum of fasting insulin fasting glucagon free fatty acids. and triglycerides,Establishment of obesity model, The 49 rats were randomly assigned to one of the following Serum FINS and FGLC concentrations were measured using. five groups 1 normal control NC n 10 2 normal diet commercial enzyme linked immunosorbent kits Shanghai Xitang. BBR NC BBR 200 mg kg day n 9 3 high fat diet HF Biotechnology Shanghai China following the manufacturer s. n 10 4 high fat diet BBR1 HF BBR1 100 mg kg day instructions Serum FFA concentrations were measured by. n 9 5 high fat diet BBR2 HF BBR2 200 mg kg day colorimetry using a commercially available kit Mike Technology. n 11 Rats in the NC group were fed regular chow and were Sichuan China following the manufacturer s instructions Serum. gavaged daily with 2 4 mL kg distilled water Rats in the TG concentrations were measured using the GPO PAP method. NC BBR group were fed regular chow per rats in the NC with a commercially available kit Mike Technology following. group and were gavaged daily with 200 mg kg BBR Rats in the the manufacturer s instructions. HF group were fed a high fat diet and were gavaged daily with. 2 4 mL kg distilled water Rats in the HF BBR1 were fed a high 2 Immunohistochemistry. fat diet per rats in the HF group and were gavaged daily with. 100 mg kg BBR Rats in the HF BBR2 were fed a high fat diet Five samples of pancreatic tissue and liver tissue were taken. per rats in the HF group and were gavaged daily with 200 from each group for immunohistochemical staining After. mg kg BBR Rats were fed their respective diets for 22 weeks fixation dehydration and clearing samples were embedded in. Body weight BW was measured once every three days and the paraffin Consecutive slices of the same sample were used for. dose of berberine was adjusted according to BW the detection of insulin IR and IRS 1 in pancreatic cells. The dose of berberine was chosen with reference to previous glucagon IR IRS 1 and IRS 2 in pancreatic cells and IR and. studies which have reported optimal glucose lowering effects IRS 1 in hepatocytes. with 100 mg kg and 200 mg kg of BBR 24 25 The tissue slices were incubated with rabbit anti mouse insulin. Berberine hydrochloride 99 5 was purchased from Hongyi polyclonal antibody 1 200 Santa Cruz Biotechnology Santa. Bio Engineering Co Ltd Sichuan China Regular chow Cruz CA rabbit anti mouse IR polyclonal antibody 1 200. contained 20 protein 5 fat 54 5 carbohydrate 7 crude ash Beijing Biosynthesis Biotechnology Beijing China rabbit anti. mainly containing minerals oxides or salts 4 5 crude fiber mouse IRS 1 polyclonal antibody 1 100 Signalway Antibody. and 9 water Chengdu Huashuo Animal Chengdu China Pearland TX rabbit glucagon monoclonal antibody 1 2000. Abcam Plc Cambridge UK or rabbit IRS 2 polyclonal antibody higher in the HF group compared with the NC group P 0 01. 1 100 Beijing Biosynthesis Biotechnology at 37 C for 45 min whereas there was no significant difference in BW between the. A universal rabbit mouse secondary antibody Dako Carpinteria NC and NC BBR groups At Week 22 BW was significantly. CA was used A DAB substrate solution was then added lower in the HF BBR1 and HF BBR2 groups compared with the. Pancreatic cells were identified by immunohistochemical double HF group P 0 01. staining whereby samples were incubated with an alkaline. phosphatase labeled glucagon specific antibody and subsequently Metabolic and biochemical parameters. a horseradish peroxidase labeled insulin receptor specific. antibody Glucagon and insulin receptor positive cells i e cells Table 2 summarizes the metabolic and biochemical. in the peripheral islet were stained dark black in color parameters at Week 22 for each group of rats All parameters. The slides were observed under a phase contrast microscope except for FBG were significantly different among the 5 groups. Olympus IX70 Olympus Corporation Tokyo Japan For VF VF BW and HOMA IR were significantly higher in the HF. pancreatic samples 3 to 7 intact islets were selected for group compared with the NC group P 0 01 Compared with. immunohistochemical analysis Immunohistochemical images the NC group BBR alone did not significantly alter any. were analyzed using Image Pro Plus 5 0 image analysis software metabolic or biochemical parameters except for VF BW. MediaCybernetics Inc Bethesda MD Protein expression P 0 01 VF and VF BW were significantly lower in both. was quantified by assessing the integrated optical density IOD HF BBR groups compared with the HF group P 0 01 FGLC. which is the average OD the total area For each tissue slice was significantly higher in the HF group compared with the. the IOD for the particular protein was assessed in 5 different NC BBR and HF BBR1 groups P 0 01. visual fields The mean IOD for rats in the same group was taken. as the expression level for the protein of interest Immunohistochemistry. Additional liver tissue slices were stained with hematoxylin. and eosin H E using standard procedures to examine cellular 1 Pancreatic islet beta cells. There were no significant between group differences in. Statistical analysis insulin expression Table 3 In contrast both IR and IRS 1. expression was significantly lower in the HF group compared with. Data are presented as means standard deviations Normally the NC and NC BBR groups P 0 01 IR and IRS 1 expression. distributed data were compared between groups using one way was numerically higher in both HF BBR groups compared with. analysis of variance with Bonferroni adjustment HOMA IR the HF group although the between group differences were not. data were non normally distributed and were compared among significant Representative microscopic images demonstrating. groups using Kruskal Wallis test and Mann Whitney U test for brown staining for insulin IR and IRS 1 are shown in Fig 1. post hoc pair wise comparisons Statistical significance was note staining in the center of the islet indicates cells. indicated by P 0 05 The statistical significance level was. adjusted to P 0 01 0 05 5 for post hoc pair wise comparisons 2 Pancreatic islet alpha cells. All statistical analyses were performed using SPSS 18 0. statistical software SPSS Inc Chicago IL Glucagon expression was significantly higher in the HF group. compared with all other groups P 0 01 Table 3 IR expression. was significantly lower in the HF group compared with the NC. RESULTS NC BBR and HF BBR2 groups P 0 01 There were no. significant between group differences in IRS 1 and IRS 2. Body weight expression Representative microscopic images demonstrating. brown staining for glucagon IR IRS 1 and IRS 2 are shown in. Table 1 summarizes the changes in BW for each group of Fig 2 note staining along the peripheral of the islet indicates. rats during the study period At Week 22 BW was significantly cells. Table 1 Comparison of changes in body weight during the 22 week study for rats fed different diets. NC NC BBR HF HF BBR1 HF BBR2,n 10 n 9 n 10 n 9 n 11. Week 0 g 0 g 306 30 38 44 308 00 17 13 312 56 16 40 298 89 10 78 296 27 25 67 0 596. Week 4 g 4 g 375 1 56 68 405 11 31 34 390 33 29 28 394 89 22 19 391 27 28 23 0 522. Week 8 g 8 g 456 50 51 97 467 78 37 16 474 22 34 74 442 67 33 20 453 55 29 11 0 461. Week 12 g 12 g 485 7 53 70 487 11 31 94 518 67 38 41 469 67 31 25 486 64 30 82 0 134. Week 16 g 16 g 497 3 45 75 486 78 27 82 547 33 29 83b 484 89 32 90c 491 45 30 75c 0 002. Week 20 g 20 g 529 10 36 18 524 00 31 55 602 44 37 68ab 511 78 21 70c 521 91 15 17c 0 001. Week 22 g 22 g 569 30 55 34 535 22 32 94 651 89 40 86ab 530 44 50 67 c 541 82 36 04 c 0 001. Data are presented as mean standard deviation and were compared among groups by one way analysis of variance with Bonferroni. adjustment, Abbreviations NC normal control diet NC BBR normal diet berberine 200 mg kg day HF BBR1 high fat diet berberine 100.
mg kg day HF BBR2 high fat diet berberine 200 mg kg day. Indicates an overall significant difference P 0 05 a Indicates a significant difference compared with the NC group P 0 01 0 05 5. b Indicates a significant difference compared with the NC BBR group P 0 01 0 05 5 c Indicates a significant difference compared. with the HF group P 0 01 0 05 5, Table 2 Comparison of metabolic and biochemical parameters for rats fed different diets for 22 weeks. NC NC BBR HF HF BBR1 HF BBR2,Parameter P,n 10 n 9 n 10 n 9 n 11. VF g 23 19 8 42 19 86 5 01 62 73 10 64ab 36 61 9 33bc 40 36 12 66abc 0 001. VF BW 0 040 0 012 0 037 0 008 0 096 0 013ab 0 069 0 014abc 0 074 0 019abc 0 001. FGLC mmol L 382 1 67 63 296 44 55 77 524 45 144 89b 323 1 84 94c 378 54 135 9 0 001. FBG mmol L 4 6 0 68 4 88 0 54 5 18 0 55 5 03 0 69 4 77 0 62 0 274. 2hPG mmol L 6 06 0 85 5 66 0 8 8 02 0 84b 6 02 1 04 6 72 1 1 0 006. TG mmol L 1 7 0 51 1 36 0 38 1 93 0 59 1 34 0 28 1 68 0 44 0 035. FFA umol L 360 21 74 08 361 29 66 47 556 59 135 32 419 52 82 17 417 6 88 79 0 001. FINS mIU L 13 58 3 22 13 18 6 41 22 69 4 78 16 06 7 81 16 2 6 28 0 006. HOMA IR 2 79 0 77 2 83 1 28 5 29 1 44ab 3 51 1 61 3 42 1 29 0 009. Data are presented as mean standard deviation and were compared among groups by one way analysis of variance with Bonferroni. adjustment except for FGLC and HOMA IR data which were compared by Kruskal Wallis test and Mann Whitney U test for post. hoc pair wise comparisons, Abbreviations NC normal control diet NC BBR normal diet berberine 200 mg kg day HF high fat diet HF BBR1 high fat. diet berberine 100 mg kg day HF BBR2 high fat diet berberine 200 mg kg day VF visceral fat BW body weight FGLC. fasting glucagon FBG fasting blood glucose 2hPG plasma glucose at two hours after meals TG triglyceride FFA free fatty acid. FINS fasting insulin HOMA IR insulin resistance index. www jpp krakow pl J J GU F Y GAO T Y ZHAO A PRELIMINARY INVESTIGATION OF THE MECHANISMS UNDERLYING THE EFFECT OF BERBERINE IN PREVENTING HIGH FAT DIET INDUCED INSULIN RESISTANCE IN RATS Department of Endocrinology and Metabolism West China Hospital of Sichuan University Wuhou District Chengdu Sichuan China Berberine exerts insulin resistance improving effects the underlying

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