Photomodulation Of Adipose Tissue On The Regulation Of Glucose Homeostasis

Objective:Accumulating evidence have shown that transplantation of adipose tissue from normal mice into obese mice induced by high-fat feeding can improve the energy metabolism and glucose metabolism of obese mice.Adipose tissue macrophages（ATMs）are strongly associated with glucose homeostasis and insulin resistance in obese and type 2diabetic populations.Macrophages are mainly divided into two subtypes,M1 classically activated macrophages and M2 selected activated macrophages.The ratio of M1 macrophages to M2 macrophages will determine glucose metabolism and insulin sensitivity.When the proportion of M1 macrophages increases,it will lead to the secretion of pro-inflammatory cytokines,thus reducing insulin sensitivity.On the contrary,when the proportion of M2 macrophages increases,it will lead to the secretion of anti-inflammatory cytokines,thus improving insulin sensitivity.However,no studies have shown that autologous adipose tissue transplantation can alter macrophage polarity to improve systemic glucose metabolism.This study attempts to use the lab self-developed light device which have specific wavelength,activate subcutaneous white adipose tissue（sWAT）separated from high-fat diet（HFD）induced obese mice,to investigate the effects of autologous transplantation of adipose tissue on macrophage polarity,glucose metabolism and insulin sensitivity;meanwhile,to explore the effects,after specific wavelength light treatment,the autologous transplantation of adipose tissue on macrophage polarity,glucose metabolism and insulin sensitivity.Methods:1.High-fat diet-induced feeding of obese mice:High-fat and high-sugar diets were continuously fed to male C57BL/6J mice aged8 weeks to age 22 weeks（continuous feeding for 14 weeks）,and the mice were randomly divided into three groups,6 mice each group,which were light-treated group,control group and sham group,and blood glucose and body weight were regularly monitored and recorded.2.Isolation of adipose tissue,light treatment,and autotransplantation:Left inguinal WAT,weighing¹20 mg,was removed from 14‐week old male HFD mice and cut into 1–2‐mm diameter pieces,which were subcutaneously transferred into a sterile tube and subjected to photoactivation for 60 min using a HarmoneyRegena device.This device integrates monochromatic lights of three different wavelengths,including 575–595 nm（5–20 mW）,630–635 nm or 660–670 nm（10–100 mW）,and/or 510–540 nm（10–60mW）of monochromatic light for 60 min.The mice were transplanted into the deep folds of the epididymal adipose tissue on the left side of the supine position and sutured;the adipose tissues of the control group mice were directly transplanted into the deep epidermal adipose tissue of the left side of the supine position and sutured;In the sham group,mouse groin sWAT was isolated,and no light treatment and autotransplantation were performed.3.Glucose tolerance test（GTT）:after fasting for 12hours,mice in the light treatment group,control group and sham group were intraperitoneal injected with glucose solution（2g/kg）.Blood samples were collected from the tail vein at 0,30,60,90 and 120 minutes.Insulin tolerance test（ITT）:after fasting for 4 hours,mice in the light treatment group,control group and sham group were intraperitoneal injected with insulin solution（0.75IU/kg）.4.PET/CT imaging of small animals:mices were anesthetized with 1%sodium pentobarbital（5mL/kg）intraperitoneally,and 100-200 uCi ¹⁸F-FDG was injected into the tail vein.The mice were fixed on a PET/CT instrument and injected¹⁸F-FDG acquired PET/CT images in 30 minutes（80kV;500 uA;1.5-mm slice thickness;10 min per bed position）.5.Detection of Insulin Content in Plasma:After fasting the mice in the light-treated group,the control group and the sham operation group for 4 hours,the glucose solution（3mg/kg）was injected intraperitoneally,and the tail vein was administered at 0 minutes,2.5 minutes,5 minutes and 15 minutes.Blood was collected,anticoagulant was added,and plasma was obtained by centrifugation at 4°C and 10,000 r/min for 10 minutes.The ELISA kit was used to detect the insulin content in plasma;cytokines such as adiponectin,TNF-α,IL-10,and IL-1β:mice were anesthetized by intraperitoneal injection of 1%sodium pentobarbital（5 mL/kg）,blood was taken from the heart,anticoagulant was added,and plasma was obtained by centrifugation at 4°C and 10,000 r/min for 10 minutes,The ELISA kit was used to detect the content of adiponectin,TNF-α,IL-10and IL-1βin plasma.6.Determination of serum lipid content:mice were anesthetized by intraperitoneal injection of 1%sodium pentobarbital（5mL/kg）,blood was taken from the heart,left to stand for 30 minutes,centrifuged at 4°C,10,000 r/min for 10 minutes to obtain serum,then detect serum triglyceride（TG）,total cholesterol（TC）,low density lipoprotein（LDL）,high density lipoprotein（HDL）,and glycated hemoglobin（HbA1c）.7.Real-time quantitative fluorescence PCR（RT-PCR）:28 days after surgery,the supine left epididymal adipose tissue and the supine right epididymal adipose tissue were collected from mices in the light-treated group,the control group,and the sham operation group,using TRIzol kit,RNA was extracted reverse-transcribed into cDNA.M1 macrophage markers such as transforming growth factor-α（TNF-α）,monocyte chemotactic protein-1（MCP-1）,and interleukin-6（IL-6）and CD11c expression were detected,and M2 macrophage markers CD206,transforming growth factor-β1（TGF-β1）,fibrin 1（Fn1）,and interleukin-10（IL-10）were detected.8.Histological examination:28days after surgery,the epididymal adipose tissue on both sides of the mice in the sham operation group,the control group and the light treatment group were separated.Divide all adipose tissue into three parts.One was fixed in an embedding box with 4%paraformaldehyde for 24 hours for immunohistochemical detection.One was frozen in liquid nitrogen and stored frozen at-80℃,then,it was used for real-time quantitative RT-PCR experiments.One was embedded with OCT embedding agent and made into frozen sections for immunofluorescent protein detection.9.Detection of vascular endothelial growth factor-A（VEGF-A）protein content:The transplanted epididymal adipose tissue and the non-transplanted epididymal adipose tissue of mice in the light-treated group,the control group,and the sham operation group were collected and washed with 1×PBS solution.After being ground in liquid nitrogen,it was stored in a 1×PBS solution at-20°C and stored overnight,and the protein content of VEGF-A in adipose tissue was detected using an ELISA kit.10.Data analysis:Data are presented as the mean±SEM.The significance of the differences between two groups was analyzed by Student’s t test.Comparisons among three groups were performed by two-way ANOVA followed by Sheffe’s test to evaluate the significance of the differences between any two groups.A level of P<0.05 was defined as indicative of statistical significance.Results:1.Transplanted light treatment of sWAT can reduce blood glucose levels in high-fat induced mice.2.After light treatment,the volume of fat cells was significantly reduced,and the weight of sWAT mice treated with light was significantly reduced.3.ELISA kit test showed that photomodulation can significantly increase the content of VEGF and IL-10 in adipose tissue,while reducing the content of TNF-αand IL-1β.4.Real-time quantitative PCR experiments showed that after light treatment,the expression of anti-inflammatory M2 macrophage markers CD206,TGF-β1,Fn1 and IL-10 were increased significantly in adipose tissue,the expression of causing inflammation M1 macrophage markers TNF-α,MCP-1,IL-β1 and CD11c were significantly reduced.5.Immunofluorescence staining experiments showed that light treatment significantly reduced the expression of TNF-αin adipose tissue and significantly increased the expression of IL-10.6.Small animal PET/CT scan experiments showed that the basal glucose uptake in the heart of the mice which transplanted light treatment of sWAT was significantly increased.Conclusion:1.Photomodulation and autologous fat transplantation can reduce the volume of fat cells and reduce the weight of mice.2.Photomodulation can change the polarity of mouse adipose tissue macrophages,transform them from M1 macrophages to M2 macrophages,increase the secretion of anti-inflammatory cytokines,reduce the secretion of pro-inflammatory cytokines,and increase insulin sensitivity,significantly reduces blood glucose levels,thereby significantly improving glucose homeostasis in mice.3.Transplanted light treatment of sWAT can significantly increase glucose uptake in mouse hearts.