| Obesity-related insulin resistance has become the main cause of type 2 diabetes in China over the age of 40.However,the role and mechanism of obesity-related insulin resistance in the pathogenesis of early-onset type 2 diabetes(EOD)in young adults aged 18-40 years old remains unclear.Studies have suggested that adipose tissue inflammation plays a crucial role in obesity-related insulin resistance.Autophagy,as a mechanism to maintain physiological homeostasis,also plays a vital role in regulating inflammation.Therefore,studying on how adipose tissue inflammation is regulated by autophagy and leads to insulin resistance among young patients(18≤age≤40 years old)with obesity,will provide important theoretical basis for the prevention and treatment of insulin resistance and EOD.Objective:We aimed to analyze the relative importance of insulin resistance and isletsβcell dysfunction as contributing factors in EOD through a case-control study.Then,we will investigate the role and mechanism of autophagy-lysosome pathway in palmitic acid-induced adipocyte inflammation in vitro.Through further case-control study,we will analyze the correlation between adipose tissue inflammation and insulin resistance,as well as the role and mechanism of the autophagy-lysosome pathway in adipose tissue inflammation among young patients(18≤age≤40 years old)with obesity.Ultimately,the study aims to provide new insights and potential targets for the prevention and treatment of obesity-related insulin resistance and EOD.Methods:1.A case-control study was conducted,with 179 newly diagnosed early-onset type 2 diabetes(EOD)adult patients admitted to the Department of Endocrinology between January 2019 and February 2021 selected as the EOD group,and 179patients who were not diagnosed with diabetes following OGTT during the same period as the control group.Data on gender,age,height,weight,insulin,blood lipids,etc.were collected and analyzed.The insulin resistance index(HOMA-IR)and insulin secretion index(HOMA-β)was calculated and analyzed.2.Mature differentiated adipocytes were exposed to different concentration(0,0.25,0.5,1m M)palmitic acid.The autophagic flow of adipocytes was detected by WB(western blot)combined with RFP-m Wasabi-LC3 adenovirus transfection and confocal microscopy.q RT-PCR and WB were used to detect the expression of CTSB and CTSL.CTSB,CTSL activity,oxidative stress index ROS and SOD activity were detected with commercial kits.WB was used to detect the expression of autophagic regulator protein TFEB,lysosomal membrane protein LAMP2 and Beclin1(a marker for initiation stage of autophagy).q RT-PCR was used to detect inflammatory factors(CCL2,IL-6 and IL-1β),WB was used to detect IL-6 and Caspase1.The indicators of adipocytes under the intervention of different concentrations of palmitic acid were compared with the control group(palmitic acid 0m M).3.A case-control study was conducted in 51 obese patients and 12 non-obese patients(18<aged≤40).Clinical data and omental adipose tissue were collected.RNA was extracted,and CTSB,CTSL and inflammatory factors(CCL2,IL-6 and IL-1β)were qualified with q RT-PCR.The insulin resistance index(HOMA-IR)was calculated and the correlation between inflammatory factors expression in adipose tissue and HOMA-IR was analyzed.CTSB and CTSL m RNA levels of adipose tissue from the two groups were compared,and the correlation between CTSB/CTSL and inflammatory factors was analyzed.Obese and non-obese cases collected in the previous step were matched based on age and gender to obtain pairs of patients with the same age and gender.The expression of autophagy indicators(P62,LC3-II),CTSB,CTSL and inflammatory factors(CCL2,IL-6,and Caspase1)in omental adipose tissue were detected with WB.Then autophagic flow,CTSB,CTSL and inflammatory factor protein levels were compared between paired cases.Results:1.No significant differences in age and gender were found in the two groups.The body mass index(BMI),triglyceride(TG),cholesterol(TC),low density lipoprotein(LDL)in EOD group were higher,and high-density lipoprotein(HDL)was lower than those in the control group(P<0.05).Ln(HOMA-IR)and Ln(HOMA-β)were standardized to Z value,logistic regression analysis was implicated and showed that HOMA-IR was a risk factor for EOD(OR=3.166,P<0.05),and HOMA-βwas a protective factor of EOD(OR=0.012,P<0.05).HOMA-IR had a higher impact on EOD than HOMA-β.Multiple linear regression analysis of HOMA-IR showed that BMI was the influencing factor of HOMA-IR,and the equation was Ln(HOMA-IR)=0.299×BMI(R~2=0.144,P<0.05)。2.Adipocytes exposed to high concentration of palmitic acid(0.5,1m M)showed upregulated LC3-II and P62 expression,and P62 turnover analysis indicated blocked P62 degradation.RFP-m Wasabi-LC3 adenovirus transfected adipocytes also showed that the autophagic clearance was blocked.The m RNA expression of CTSB and CTSL were upregulated(P<0.05),while active CTSL(25kd)and the activity of CTSL were downregulated(P<0.05),and both of the active-CTSB(35kd)and CTSB activity were upregulated(P<0.05).Palmitic acid increased ROS level of adipocytes,inhibited SOD activity(P<0.05),increased the m RNA expression of CCL2 and IL-6,and upregulated the protein expression of IL-6 and Caspase1(10kd).Palmitic acid(0.25,0.5,1m M)increased the expression of TFEB in the nucleus(P<0.05),while no significant differences in the expression of LAMP2 and Beclin1 across the groups were found.The results suggested that adipocytes exposed to palmitic acid showed lower autophagic clearance,which was not related to LAMP2,but caused by deteriorated CTSL activity.Compensatory activation of CTSB and autophagic clearance dysfunction were involved in inflammation of adipocytes induced by palmitic acid.3.The m RNA levels of CCL2,IL-6 and IL-1βof omental adipose tissue from obese patients were upregulated and all positively correlated with HOMA-IR(r=0.353-0.457,P<0.001).The m RNA levels of CTSB and CTSL were upregulated(t=2.693,P<0.01;t=2.849,P<0.01)too,and showed strongly positive correlation with each other(r=0.836,P<0.001).Both of CTSB and CTSL m RNA levels were related to TC(stdβ=0.443,P<0.05,std.β=0.439,P<0.05),while only CTSB was associated with BMI(stdβ=0.261,P<0.05).Multiple regression analysis showed that not CTSB,but CTSL correlated with CCL2,IL-6 and IL-1βindependently(stdβ=0.352-0.462,P<0.05).It suggested that the inflammation of omental adipose tissue is positively correlated with insulin resistance,and elevated CTSL gene expression correlated with inflammatory cytokines independently in omental adipose tissue of young patients with obesity.In comparison to non-obese young patients,patients with obesity exhibited upregulation of mature CTSB protein and downregulation of mature CTSL expression in their omental adipose tissue.Additionally,autophagy indicators P62 and LC3-II were found to be increased,and the expression of inflammatory factors were elevated(P<0.05).These findings suggest that the autophagy flow was blocked and inflammatory factors were upregulated in adipose tissue of young patients with obesity.Conclusion:1.Insulin resistance,but not isletsβCell dysfunction,is a more important risk factor for EOD.Obesity is an important risk factor for insulin resistance.2.Palmitic acid impaired CTSL function and decreased CTSL enzyme activity in adipocytes,leading to lower autophagic clearance and compensatory activation of CTSB,together increased the expression of inflammatory factors of adipocyte.3.The m RNA levels of inflammatory factors(CCL2,IL-6 and IL-1β)in omental adipose tissue from obese patients were upregulated and positively correlated with HOMA-IR,indicating that adipose tissue inflammation may contribute to the development of insulin resistance in obesity.4.The gene expression of CTSL in adipose tissue is an independent influencing factor of adipose tissue inflammation in young patients with obesity.The dysfunction of the autophagy-lysosome pathway,associated with CTSL and CTSB,was identified as an important contributor to obesity-related inflammation in young patients with obesity. |
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