| Part 1:Improvement of Energy Metabolism by Regulating Brown Adipose function of CB1 Cannabinoid ReceptorBackground and aims:The mechanisms of obesity have not been fully clarified. There has been lot of studies by enhance the BAT function in the treatment of obesity. Endocannabinoid system closely related to obesity and insulin resistance. However, the endocannabinoid system can through the enhancement of brown fat function and reduce obesity is unclear. In this study, the effect of CB1 receptor on the function of brown fat,glucose homeostasis,insulin sensitivity,food intake and energy metabolism were observed by CB1 knockout mouse model.Materials and methods:WT mice and CB1-/- mice were housed under specific pathogen free facility. Mice fat mass and lean mass were analyzed by EchoMRI Whole Body Composition Analyzer every 2-4 week. The insulin tolerance tests and the glucose tolerance tests were carried out on WT mice and CB1-/- mice. The mice tail blood glucose and blood insulin level was measured.Metabolic parameters (food intake, locomotor activity, energy expenditure (or heat) were measured using an Oxymax open-circuit indirect calorimetry system. The H&E-stained slides of scapular brown adipose tissue were analyzed and the brown cell size were compared. The expression of genes associated with energy metabolism (UCP-1.PGC-la) and lipid metabolism(IR、IRS1、PPARγ2) in the brown fat were assayed by realtime PCR and Western blotting. The intraperitoneal inflammation index of WT mice and CB1 knockout mice group was detected by flow cytometry instrument.Results:It was showed that CB1-/- mice showed that a consistent reduction of body weight in the CB1-/－ mice compared with their WT littermates from 10 weeks- to 34 weeks. During GTT,CB1-/- mice group (Omin 12718, 15min 481+21, 30min 459±33, 60min 409±37, 120min 191±24mg/dl)was similar to WT mice group (Omin 123±12, 15min427±26, 30min436±16,60min 4041±33, 120min 209±28mg/dl)in glucose clearance. And the insulin level of the CB1-/- mice group was significantly lower(WT mice: Omin 1.46±0.15, 15min 2.09±0.34, 30min 1.88±0.19,120min 2.68±0.46ng/ml ;CB1-/- mice:0minl.07±0.07 , 15min 1.2810.17 ,30min1.30±0.15 , 120min1.66±0.11ng/ml),which was the indicative of increased insulin sensitivity(P<0.05).During ITT, CB1-/- mice (0minl66±4, 30min 96±5 , 60min88±6,90minl 11±10,120min153113 mg/dl)were significantly more sensitive to the insulin challenge compared to WT mice(0min 181±3, 30min l37±11, 60min135±13,90min142±16, 120min 173±21mg/dl) (P<0.05).The daily energy intake (food intake) , the locomotor activity and respiratory quotient (RQ) of WT and CB1-/- mice was comparable during indirect calorimetry(P>0.05).CB1-/- mice have significantly higher energy expenditure compared with WT mice(P<0.05).Histological analysis of brown fat showed that CBl-/- mice have smaller adipocytes(853.2± 109.5 vs 693.4 ± 148.5μm2). Quantitative analysis of the size of brown adipocytes indicated that CB1-/- mice had more small adipocytes(P<0.05).The mRNA and protein expression of genes (UCP-1、PGC-1 a、IR.IRS 1、PPARy2) associated with thermogenesis and lipid metabolism in the brown fat of CB1-/－ mice significantly increased compared with WT mice. These results suggested that CB1-/- mice improved the capacity of converting glucose and fat into heat production (P<0.05).The results of flow cytometry showed that M1 decreased, M2 increased and ratio of M1/M2 decreased in the peritoneal macrophages of CB1-/- mice(P<0.05).Conclusion:Body weight and body fat of CB1-/- mice become lighter compared with that of WT mice,while the insulin sensitivity of CB1 -/- mice become more sensitive, compared with that of WT mice. Although feeding pattern has changed, there is no difference in the food intake and locomotor activity between WT mice group and CB1-/- mice group. In addition, the metabolic analysis demonstrated that CB1-/- mice increased energy expenditure throughout the light and dark phases,and the mRNA and protein expression of genes associated with thermogenesis and lipid metabolism in the brown fat of CB1-/- mice increased. The mechanism of weight loss in CB1 receptor knockout mice may be by enhancing the function of brown fat.Part 2 GLP-1 receptor agonist improves function of brown adipose tissue in mice with diet-induced obesityBackground and aims:Recent studies have indicated that GLP-1 receptor (GLP-1 R) agonist has shown favorable effects on lowering blood sugar and its effects on body weight reduction in rat and human studies.To investigate the benefits of GLP-1 receptor agonist ,exendin-4 treatement on body weight ,brown fat function ,glycometabolism ;lipid metabolism, food intake and energy metabolism in C57BL/6J mice induced with high-fat diet.Materials and methods:The six-week-old C57BL/6J mice were randomly divided into 3 groups, including control group, high-fat diet (HFD) group, and HFD group treated with exendin-4. Mice fat mass and lean mass were analyzed by EchoMRI Whole Body Composition Analyzer every 2-4 week.The insulin tolerance tests and the glucose tolerance tests were carried out on three group after 16 weeks. Metabolic parameters (food intake, locomotor activity energy expenditure )were measured using an Oxymax open-circuit indirect calorimetry system between HFD group and the exendin-4 treated group. The scapular brown adipose tissue were analyzed with H&E staining. And the brown cell size were measured among three group. The expression of genes associated with energy metabolism and lipid metabolism in the brown fat were tested with realtime PCR and Western blotting.Results:With increasing time, the final body weight and fat mass of the HFD group was higher than that of the control group, but body weight and fat mass of the exendin-4 treated group was lower than that of the HFD group (P<0.05).During GTT, HFD group showed slower glucose clearance compared with CON group, but the glucose clearance of group treated with exendin-4 was faster than that of HFD group (0-30-60-90-120min: CON 组:91±4-359±50-345±35-260140-169±33mg/dl vs.HFD 组:122117-430±55-490±38-453±76-228147mg/dl vs.HFD+EX 组 116±16-373±49-391±55-326±57-182±34 mg/dl) (P<0.05). During ITT, HFD group became severely insulin resistant. Remarkably, exendin-4 treated group were more sensitive to the insulin challenge compared to HFD group (CON:119±11-68±17-84±8-91±8-101±1lmg/dl vs.HFD:13418-113±17-10216-115±5-120±4 mg/dl vs.HFD+EX:123±11－93±12-89±27-96±19-106±13 mg/dl) (P<0.05), which indicated that exendin-4 could improve insulin sensitivity. There were no significant difference in the daily energy intake (food intake) and the locomotor activity between the HFD group and the exendin-4 treated group during indirect calorimetry experiment (P>0.05).Exendin-4 treated group have significantly higher energy expenditure when compared with compared with HFD group(P<0.05).Furthermore, our data demonstrated that exendin-4 treated group have a decreased respiratory quotient (RQ) compared with HFD group(P<0.05).Histological analysis of brown fat that HFD group have significantly greater adipocytes when compared with CON group(3011.8 ± 566.3 vs 711.3 ±247.1 μm2), exendin-4 treated group had more small adipocytes(956.8 ±350.1 μm2) (P<0.05). The mRNA and protein expression of genes associated with thermogenesis and lipid metabolism in the brown fat of HFD group significantly decreased compared with CON group, as expected, the mRNA and protein expression of genes associated with thermogenesis and lipid metabolism were increased in the brown fat of exendin-4 treated group when compared to that of HFD group(P<0.05).Conclusion:GLP-1 agonist (exendin-4) treatment reduced high-fat induced obesity by decreasing body weight and adiposity, improving impaired glucose tolerance and decreased insulin sensitivity,and improving lipid metabolism. During indirect calorimetry exendin-4 treatment can increase energy expenditure and the mRNA and protein expression of genes associated with thermogenesis and lipid metabolism in the brown fat. These results suggest GLP-1 agonist may be a new means of combating obesity by shifting the energy balance from obesogenesis to thermogenesis.Part 3 Exendin-4 enhances the functions of brown adipocytes by antagonizing CB1 receptorBackground and aims:Brown adipocytes were cultured in vitro. The aim was to study whether exendin-4(a GLP-1 receptor agonist) can impact the functions of brown adipocytes by antagonizing CB1 receptor, and to investigate its downstream signaling pathway.Materials and methods:The expression of CB1 receptor of brown adipocytes in different concentrations of GLP-1 receptor agonists (exendin-4) and GLP-1 receptor antagonist exendin (9-39) were assayed by real-time PCR. The expression of genes associated with energy metabolism (UCP-1、PGC-la)and lipid metabolism(PPARy2) in the brown adipocytes were assayed by real-time PCR under the intervention of the GLP-1 receptor agonist exendin-4 and CB1 receptor agonist WIN55.The energy metabolism of brown adipocytes was measured by Seahorse under the intervention of the GLP-1 receptor agonist exendin-4 and CB1 receptor agonist WIN55. The expression of protein associated with signaling pathway (AMPK) in brown adipocytes were assayed by Western blotting under the intervention of GLP-1 receptor agonist exendin-4 and CB1 receptor agonist WIN55.Results:GLP-1 receptor agonist exendin-4 reduced the expression of CB1 receptor in a concentration dependent manner, whereas the GLP-1 receptor antagonist exendin (9-39)increased the expression of CB1 receptor in the brown adipocytes (P<0.05).The expression of genes associated with thermogenesis (UCP-1、PGC-la) and lipid metabolism (PPARy2)decreased in the brown adipocytes of WIN55 intervention group, compared to that of CON group; and the mRNA expression of UCP-1、 PGC-la and PPARy2 in the brown adipocytes of exendin-4 intervention group significantly increased compared to CON group(P<0.05);however, the expression of UCP-1、PGC-la and PPARy2 were significantly decreased in exendin-4 combined with WIN55 group than that of exendin-4 intervention group(P<0.05). The maximum respiratory intensity in brown adipocytes was significantly increased in exendin-4 group than that of CON group, and the maximal respiratory intensity was decreased in WIN55 intervention group compared with CON group (P<0.05). The maximal respiratory intensity of brown adipocytes was significantly decreased in exendin-4 combined with WIN55 group than that of exendin-4 intervention group individual (P<0.05). The protein expression of pAMPK in brown adipocytes was significantly increased in exendin-4 group than that of CON group,and the protein expression of pAMPK in the brown adipocytes of exendin-4 group was significantly increased compared with CON group. The protein expression of pAMPK in brown adipocytes was significantly lower in exendin-4 combined with WIN55 group than that of exendin-4 intervention group individual.Conclusion:Exendin-4 may affect the expression of downstream genes associated with energy metabolism and lipid metabolism in the brown adipocytes by blockade of brown adipocytes of CB1 receptor, and play an important role in activating the AMPK signaling pathway. The research further clarified the mechanism of GLP-1 of enhancing the function of brown fat, and extended new thinking for the treatment of obesity. |
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