| Objective:In clinical work,it is found that obese male patients are often accompanied by hypoandrogenemia,but there is no large sample clinical study on the correlation between obesity and male sex hormones,and there is no consistent conclusion on whether obese male patients with testosterone deficiency need testosterone replacement therapy.The effect and mechanism of testosterone on metabolism in obese male patients remain to be clarified.Therefore,the purpose of this study is to comprehensively analyze the relationship between male sex hormones and obesity and related metabolic indexes,to systematically evaluate the efficacy and safety of testosterone replacement therapy on metabolism in obese male patients with testosterone deficiency,and to explore the effects of testosterone deficiency and testosterone replacement therapy on metabolism in male obese rats.So as to provide clinical,evidence-based,animal and molecular biological basis for clinical decision-making of testosterone replacement therapy in obese male patients with testosterone deficiency.Materials and Methods:1.Retrospective study partAccording to the inclusion and exclusion criteria,this study retrospect-tively analyzed the data of adult male patients(20-60 years old)hospitalized in the Endocrinology Department of West China Hospital.body mass index(BMI),waist circumference(WC),sex hormone binding globulin(SHBG),total testosterone(TT),estradiol(E2),luteinizing hormone(LH),follicle stimulating hormone(FSH),blood lipid,fasting blood glucose(GLU)and serum albumin(Alb)were collected.The values of free testosterone(FT)and Bioactive testosterone(Bio T)were calculated by TT,SHBG and Alb.The patients were divided into subgroups according to BMI and WC,and the differences of sex hormone indexes(SHBG,TT,FT,Bio T,E2,LH,FSH)among different BMI and WC groups were analyzed.Multivariate linear regression was used to analyze the correlation between sex hormone indexes and BMI,WC,age,glucose as well as lipid.2.Systematic review partRandomized controlled trials of male obese patients with testosterone deficiency treated by testosterone replacement therapy(TRT)in Cochrane Library,Pub Med,Embase,Web of Science,Scopus database,Open SIGLE database,CNKI,Wanfang,VIP,CBM Database were retrieved by computer.The literatures were screened according to the inclusion and exclusion criteria.Rev Man 5.3 bias risk assessment tool(Risk of bias)and Jadad score were used to evaluate the methodological quality of randomized controlled trials.Extract data according to Cochrane system Evaluation Manual 5.1.The effects of testosterone replacement therapy on obesity and metabolic indexes were evaluated by quantitative Meta analysis or qualitative system.The outcome indexes of more than 3 articles were analyzed by Meta with Rev Man 5.3 software.I~2value was used to test the heterogeneity among studies,and 0.05 was used as the test level.The sensitivity analysis was carried out by eliminating individual studies one by one,and the publication bias was analyzed by Egger test.3.Animal experiment partMale 5-week-old SD rats were fed with normal diet(NC group)and high-fat diet(obesity group)for 15 weeks.The obese model rats were divided into obese control group(OC group),obese central castrated group(OCC group,testosterone deficiency caused by leuprorelin central castration for 10 weeks)and obese with testosterone replacement after castration group(OCT group,central castration caused by leuprorelin for 10 weeks.From the 4th week,testosterone undecanoate was injected intramuscularly).The body weight of rats was measured every week during the intervention period.After the intervention,glucose tolerance test and insulin tolerance test were performed,and lee’s index,fasting blood glucose,abdominal fat content,skeletal muscle content,liver weight,plasma insulin,plasma testosterone and blood lipids were detected.The cross-sectional area and diameter of skeletal muscle fibers were detected by HE staining,the degree of skeletal muscle fibrosis was detected by masson staining,the apoptosis index of skeletal muscle was detected by Tunel staining,and the protein expression of AKT,P-AKT(ser473),P-AKT(thr308)and P-m TOR in skeletal muscle protein synthesis pathway was detected by Western-Blot.The expression of chromatin remodeling complex BAF60a subunit of gastrocnemius was detected by immunofluorescence.QRT-PCR and Western-Blot were used to determine the expression of mitochondrial oxidative metabolism regulation indexes(BAF60a,PPARα,PGC-1α),mitochondrial oxidative metabolism indexes(m GPDH,UCP2),and energy metabolism substrate transporters(FAT/CD36,GLUT4)in gastrocnemius muscle.Results:1.Retrospective study part1)A total of 360 male patients were included in this study.the average age was 44.23±10.95 years old,and the average level of BMI was 25.21±4.18Kg/m~2.2)According to BMI,patients were divided into three groups:normal group(18.5≤BMI<24Kg/m~2group),overweight group(24≤BMI<28Kg/m~2group)and obese group(BMI≥28 Kg/m~2group).The results showed that:1)SHBG(nmol/L):obese group(27.35±18.02)and overweight group(31.47±15.54)were less than normal group(41.23±18.35),P<0.05).2)TT(ng/ml):the levels of TT in obesity group,overweight group and normal group decreased in turn(P<0.05).There was no significant difference in the levels of other sex hormones(FT,Bio T,E2,LH,FSH)among different BMI groups.3)Taking BMI≥33 Kg/m~2as the cut-off point,the obese group was further divided into obese group 1(28≤BMI<33 Kg/m~2group)and obese group 2(BMI≥33 Kg/m~2group).The difference of free testosterone(FT)was found between groups.The FT(ng/ml)in obesity group2(0.064±0.034)was lower than that in obesity group 1(0.085±0.027),overweight group(0.091±0.027)and normal group(0.090±0.036),P<0.05.4)The patients were divided into two groups according to their waist circumference:normal group(WC<90cm)and abdominal obesity group(WC≥90 cm).It was found that SHBG(30.33±16.82 vs.39.87±18.34nmol/L)and TT(4.06±1.62 vs.4.06±1.62 ng/ml)in abdominal obesity group were lower than those in normal group,and there was no significant difference in other sex hormones(FT,Bio T,E2,LH,FSH)between the two groups.5)Multiple linear regression was performed with sex hormone indexes as dependent variables and BMI,waist circumference,age,blood gluose and triglyceride as independent variables.In comprehensive analysis,it is found that,SHBG was negatively correlated with BMI,waist circumference,triglyceride,and positively correlated with age;total testosterone was negatively correlated with BMI,waist circumference and blood glucose;free testosterone was negatively correlated with BMI,waist circumference,age and blood glucose;bioactive testosterone was negatively correlated with BMI,waist circumference,age and blood glucose;estradiol was positively correlated with BMI and negatively correlated with triglyceride.Luteinizing hormone and follicle stimulating hormone were only positively correlated with age.2.Systematic review partA total of 3 randomized controlled trials were included in the study.One literature was medium quality literature and two were high quality literature.All of the literature were published in English language.A total of 201 patients were included in the baseline.Compared with the control group,testosterone replacement therapy(TRT)had no significant improvement in body mass index(MD=-0.10,95%CI:-0.61～0.40,P=0.69)and waist circumference(MD=-0.49,95%CI:-1.25～0.27,P=0.21)in obese male patients with testosterone deficiency,but could improve body composition,reduce total body fat,increase lean body mass or reduce lean body weight loss after energy intake restriction.The effects of TRT on fasting blood glucose,glycosylated hemoglobin and insulin sensitivity are not consistent.TRT had no significant effect on triglyceride(MD=-0.19,95%CI:-0.44～0.06,P=0.13),total cholesterol(MD=-0.20,95%CI:-0.4～0.00,P=0.05),high density lipoprotein cholesterol(MD=-0.01,95%CI:-0.08～0.06,P=0.71)and low density lipoprotein cholesterol(MD=0.07,95%CI:-015～0.29,P=0.52).TRT also had no significant effect on systolic blood pressure and diastolic blood pressure.In the three studies,hemoglobin(Hb)and hematocrit(Hct)both increased in TRT group.The level of prostate specific antigen(PSA)in TRT group increased in two studies.3.Animal experiment part1)The body weight of rats in the obesity group was significantly higher than that in the normal control group.However,there was no significant difference in body weight among obesity subgroups(OC group,OCC group and OCT group).2)There was no significant difference in serum testosterone level between OC group and NC group(98.33±13.22 vs.90.63±16.51 pg/ml,P>0.05).Serum testosterone(12.31±0.76pg/ml)in OCC group was significantly lower than that in NC group,OC group and OCT group.The level of serum testosterone in OCT group(31.07±3.01pg/ml)was higher than that in OCC group,but lower than that in OC group and NC group.It is suggested that after central castration,the rat model of obesity with testosterone deficiency was successful,the level of serum testosterone increases after testosterone intervention,and testosterone intervention was effective.3)The area under glucose curve in oral glucose tolerance test and insulin tolerance test,fasting blood glucose,insulin level and insulin resistance index(HOMA-IR)in obesity group were higher than those in NC group,but there was no significant difference among OC group,OCC group and OCT group.4)There was no significant difference in blood lipid indexes(triglyceride,total cholesterol,low density lipoprotein cholesterol,high density lipoprotein cholesterol,very low density lipoprotein cholesterol)among NC group,OC group,OCC group and OCT group.5)The total skeletal muscle weight in OCC group was lower than that in OC group and OCT group(P<0.05).The skeletal muscle-to-body weight ratio in OC group,OCC group and OCT group was significantly lower than that in NC group(1.30±0.06%、1.21±0.04%、1.29±0.05%vs.1.63±0.08%,P<0.05).The skeletal muscle-to-body weight ratio in OCC group was significantly lower than that in OC group(1.21±0.04 vs.1.30±0.06%,P<0.05).6)Compared with NC group,perirenal fat,epididymal fat,mesenteric fat,abdominal total fat and abdominal fat body weight ratio of OC,OCC and OCT groups were higher(P<0.05).Comparison between obese groups,mesenteric fat,total abdominal fat and abdominal fat-to-body weight ratio of OCC group was higher than that of OC group(15.41±0.83g vs.11.94±2.75g,60.86±3.43g vs.54.95±6.64,9.41±0.58%vs.8.53±0.91%,P<0.05).7)The liver weight of obese rats was significantly higher than that of NC group,but there was no significant difference among the three groups(OC,OCC,OCT groups).8)HE staining showed that the cross-sectional area and diameter of skeletal muscle in OCC group and OCT group were significantly lower than those in NC group(P<0.05),but there was no significant difference among obese groups(OC group,OCC group,OCT group).9)Masson staining of skeletal muscle showed that the phenomenon of skeletal muscle fibrosis was significantly increased in OCC group,and the degree of skeletal muscle fibrosis in OCT group was less than that in OCC group.10)Tunel staining of skeletal muscle showed that the apoptotic index of skeletal muscle in OC group was 3.69 times higher than that in NC group.In OCC group,the apoptotic index was 1.9 times higher than that in OC group after castration.In OCT group,the apoptotic index was 24%lower than that in OCC group after testosterone replacement therapy.11)Protein expression of skeletal muscle protein synthesis pathway by Wester-Blot showed that the change trend of P-AKT(ser473),P-AKT(thr308)and P-m TOR were consistent.in OC group,the expression of P-AKT(ser473),P-AKT(thr308)and P-m TOR was 41.90%,37.52%and55.95%lower than that in NC group.In OCC group,P-AKT(ser473),P-AKT(thr308)and P-m TOR Protein expression were 50.79%,62.5%and58.10%lower than that in OC group,respectively.After testosterone replacement therapy,in OCT group,those indexs increased by 80.04%,73.88%and 129.03%compared with the OCC group.There was no significant difference in the protein expression of AKT among the four groups.4.Skeletal muscle energy metabolism part1)Immunofluorescence staining of skeletal muscle showed that the expression of BAF60a protein in OC group was lower than that in NC group(P>0.05).The expression of BAF60a in OCC group was 36.5%lower than that in NC group(P<0.05)and 31.3%lower than that in OC group(P<0.05).Compared with the OCC group,the expression of BAF60a in OCT group recovered and increased by 45%after testosterone replacement therapy(P<0.05).2)QRT-PCR results showed that the change trend of m RNA expression of BAF60a,PPARαand PGC-1αgenes was consistent among groups.Compared with NC group,the m RNA expression of BAF60a,PPARαand PGC-1αin OC group decreased by 40%,30%and 36.2%respectively(P<0.05).Compared with OC group,the m RNA expression of BAF60a,PPARαand PGC-1αin OCC group decreased by 38.9%,32.8%and 26.4%respectively(P<0.05).Compared with OCC group,the expression of BAF60a,PPARαand PGC-1αin OCT group increased by50%(P<0.05),42.2%(P<0.05)and 30.2%(P>0.05)respectively.3)The results of Western-Blot showed that the change trend of BAF60a,PPARαand PGC-1αprotein expression was consistent among the groups.Compared with NC group,the protein expression of BAF60a,PPARαand PGC-1αdecreased significantly by 20.3%,40.2%and 31.4%respectively(P<0.05)in OC group.Compared with OC group,the protein expression of BAF60a,PPARαand PGC-1αdecreased by 34.8%,55.4%and 53.7%respectively(P<0.05)in OCC group.Compared with OCC group,the protein expression of BAF60a,PPARαand PGC-1αincreased by 29.1%,74.4%(P<0.05)and 119.2%(P<0.05)respectively in OCT group.4)The results of q RT-PCR and Western-Blot showed that the change trend of m GPDH m RNA level and protein expression were consistent among the groups.Compared with NC group,m GPDH m RNA level and protein expression were significantly decreased by 40.3%and 40.2%respectively in OC group(P<0.05).Compared with OC group,m GPDH m RNA level and protein expression decreased by 30.6%and 65.8%respectively in OCC group(P<0.05).Compared with OCC group,m GPDH m RNA level and protein expression increased by 34.2%and 93.6%respectively in OCT group(P<0.05).5)The results of Western-Blot showed that compared with NC group,the expression of UCP2 protein decreased by 40.9%in OC group(P<0.05).Compared with OC group,the expression of UCP2 protein further decreased by 64.9%in OCC group(P<0.05).Compared with OCC group,the expression of UCP2 protein increased in OCT group.6)The results of Western-Blot showed that the expression of glucose transporter GLUT4 and fatty acid transporter FAT/CD36 in skeletal muscle cells had the opposite trend.Compared with NC group,the GLUT4expression decreased by 27.4%in OC group(P<0.05).Compared with the OC group,the GLUT4 protein expression further decreased by 38.3%in OCC group(P<0.05).Compared with OCC group,the GLUT4 protein expression increased by 42.3%in the OCT group(P<0.05).The change trend of FAT/CD36 protein expression between groups was opposite to that of GLUT4.Compared with NC group,FAT/CD36 protein expression increased by 79.4%in OC group(P<0.05).Compared with OC group,it increased by 37.3%in OCC group(P<0.05).And it decreased by 15.8%in OCT group when compared with OCC group(P<0.05).Conclusion:1.The sex hormone binding protein(SHBG)and total testosterone(TT)of obese group were lower than that of normal weight group.The free testosterone in severely obese patients with BMI≥33 Kg/m~2was also lower than that in patients with normal body weight.SHBG was negatively correlated with BMI,waist circumference,triglyceride,and positively correlated with age.Total testosterone was negatively correlated with BMI,waist circumference and blood glucose.Free testosterone was negatively correlated with BMI,waist circumference,age and blood glucose.2.The systematic evaluation of randomized controlled trials showed that testosterone replacement therapy in obese male patients with testosterone deficiency could reduce body fat,increase lean body weight and improve body composition,but had no significant effect on body mass index,waist circumference,body weight,blood lipid and blood pressure.There is a risk of increased hemoglobin,hematocrit and prostate specific antigen.3.In obese rats,the skeletal muscle-to-body weight ratio decreased and the abdominal fat-to-body weight ratio,blood glucose,HOMA-IR,apoptosis index of skeletal muscle increased.The protein expression of P-AKT(ser473),P-AKT(thr308)and P-m TOR in skeletal muscle decreased.Testosterone deficiency further reduced the skeletal muscle-to-body weight ratio and increased the abdominal fat-to-body weight ratio in obese male rats,which further worsened sarcopenic obesity.Skeletal muscle fibrosis and skeletal muscle cell apoptosis were further aggravated,the expression of P-AKT(ser473),P-AKT(thr308)and P-m TOR protein in protein synthesis pathway was further decreased.Testosterone replacement therapy can improve the above phenomenon to some extent.The mechanism of the decrease of skeletal muscle content in obese rats with testosterone deficiency is related to the decrease of protein synthesis and the increase of apoptosis in skeletal muscle.The decrease of testosterone level and testosterone replacement therapy had no significant effect on body weight,glucose and lipid metabolism in obese male rats.4.The oxidative metabolism of skeletal muscle mitochondria in obese rats was impaired.The regulatory indexes of fatty acid oxidative metabolism(BAF60a,PPARα,PGC-1α),mitochondrial oxidative metabolism indexes(m GPDH,UCP2)and the expression of glucose transporter GLUT4 decreased,while FAT/CD36 increased.The level of testosterone affects the expression of key indexes of energy metabolism in skeletal muscle mitochondria of obese rats.The level of testosterone decreased,the expression of BAF60a,PPARα,PGC-1α,m GPDH,UCP2,GLUT4 further decreased,and the expression of FAT/CD36 protein further increased,which was improved after testosterone replacement therapy.It was suggested that obesity and testosterone levels could affect the expression of key indexes of mitochondrial oxidation metabolism and affect the energy metabolism of skeletal muscle in male rats.Testosterone deficiency could aggravate the disturbance of oxidative metabolism of skeletal muscle mitochondria,aggravate apoptosis and protein synthesis disturbance of skeletal muscle,further reduce the content of skeletal muscle,and then worsen sarcopenic obesity in male rats.Testosterone replacement therapy could partially reverse this pathological state. |
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