in the past 30 years , the global prevalence of obesity has increased among all age groups . obesity leads not only to an increase in adipose tissue mass but also to the infiltration of proinflammatory cells and secretion of inflammatory cytokines [ 1 , 2 ] . therefore , obesity is characterized by low - grade inflammation in local and systemic sites as demonstrated by robust secretion of proinflammatory cytokines , including il-6 , as well as active recruitment of leukocytes . substantial evidence supports the hypothesis indicating that inflammation may contribute to insulin resistance , which further induces a series of diseases such as diabetes , hypertension , fatty liver disease , and coronary heart disease , thereby threatening human health [ 4 , 5 ] . autophagy includes three basic forms , namely , macroautophagy , microautophagy , and chaperone - mediated autophagy ( cma ) . macroautophagy ( henceforth termed autophagy ) is a lysosomal degradation pathway , which can degrade the organelles , longevity protein , and lipid drops and thus provide energy for the body [ 7 , 8 ] . when the body faces various pressures caused by acute stress , autophagy plays a key role in maintaining the stability of the internal environment , particularly in regulating apoptosis and resisting the invasion of pathogenic microorganisms . self - renew , repair , and differentiation of cells are important for metabolism and maintenance of energy balance . studies have shown that autophagic dysfunction is closely related to metabolic disorders , such as insulin resistance , diabetes , obesity , and osteoporosis . high mobility group box 1 protein ( hmgb1 ) is a nonhistone nuclear factor and a highly conserved protein . hmgb1 can bind to chromosomal dna to adjust the refactoring of chromatin [ 11 , 12 ] . hmgb1 is abundant in the vast majority of mammalian cells   and plays a key role as a signal molecule extracellularly . hmgb1 can be passively released from necrotic cells or actively secreted from inflammatory cells [ 15 , 16 ] . aseptic injury to cells increases the level of hmgb1 in serum and tissues . as such , hmgb1 is associated with low - grade inflammation diseases , such as obesity and type 2 diabetes . some research found that hmgb1 interacted with autophagy through its different receptors , outside the cells by receptor of advanced glycation end products ( rage ) , within the nucleus through heat shock protein beta-1 ( hspb1 ) , and within the cytoplasm through becn1 . however , little is known about how hmgb1 , autophagy , and adipocytes interact to regulate adipocyte development and differentiation . the present research mainly focused on the effects of hmgb1 on autophagy and cell differentiation in adipocytes . antibodies were obtained from the following sources : hmgb1 and gapdh from abcam , lc3 from cell signaling , and p62 from proteintech group . the following reagents were purchased from sigma : 1-methyl-3-isobutylxanthine , dexamethasone , insulin , oil - red - o dye , and hematoxylin and eosin . rearing environment indoor temperature was controlled at 20c to 25c , relative humidity within 40% to 60% , lights 12  h every day , along with free drinking water in the cage . six - week - old male mice were randomly divided into two groups , namely , the normal - diet ( nd ) group and high - fat diet ( hfd ) group , with 10 mice in each group . mice were fed correspondingly with standard chow ( 10% kcal in fat ) or hfd ( 45% kcal in fat ) for 16 weeks . mouse epididymal adipose tissue samples were fixed in 4% paraformaldehyde and paraffin - embedded , then cut into 4  m thick sections , and deparaffinized in xylene and rehydrated in a descending ethanol series . the sections were stained with hematoxylin and eosin using standard pathologic procedures . finally , photomicrographs of all sections were taken by leica dm500 microscope . 3t3-l1 preadipocytes were maintained and cultured in dulbecco 's modified eagle 's medium ( dmem ) with existing 10% heat inactivated fetal bovine serum and 1% penicillin / streptomycin at 37c in 5% co2 . 3t3-l1 preadipocytes were first cultured in a medium supplemented with 10  g / ml insulin , 1  m dexamethasone , and 0.5  mm 1-methyl-3-isobutylxanthine for 2 days . differentiated adipocytes were cultured in dmem containing 10% fetal bovine serum [ 1 , 20 ] . to invest the effects of hmgb1 protein on autophagy in various differentiation periods of 3t3-l1 adipocytes , the adipocytes were treated with hmgb1 protein 24  h before differentiation . in detail , the 3t3-l1 preadipocytes were induced at 0 , 2 , 4 , 6 , 8 , and 10 days , and the 0.2  g / ml recombinant hmgb1 proteins were added at days 1 , 1 , 3 , 5 , 7 , and 9 and removed at days 0 , 2 , 4 , 6 , 8 , and 10 . 3t3-l1 preadipocytes were grown to approximately 50% to 70% confluence in a six - well plate and then transfected with 75  pmol hmgb1 sirna and control sirna by using lipofectamine 2000 in accordance with the manufacturer 's instructions . after transfection for 6  h , the medium was replaced with a normal medium . differentiated adipocytes were stained by oil - red - o , as follows : 0.5  g oil - red - o powder was dissolved in 100  ml of isopropanol overnight . the solution was filtered and stored in a brown bottle at 4c . after the medium was discarded , cells were then washed with pbs and stained with the oil - red - o dye solution for 1  h. subsequently , cells were washed with pbs and treated with 60% isopropanol for 5  min . finally , cells were washed and photographed under a microscope . 3t3-l1 cells were collected and lysed in ice - cold lysis buffer ( 5.0  mm tris buffer , ph 7.4 , 150  mm nacl , 1% np-40 , 0.1% sds , 0.5% deoxycholic acid , 1  mm edta , 1  mm pmsf , 2  g/l aprotinin , 2  g/l leupeptin , and 1  mm naf ) for 40  min and then subsequently boiled and quantified . after the samples were resolved by sds - page for 2  h , proteins were transferred to nitrocellulose membranes at 200  v for 90  min . membranes were blocked in 5% nonfat milk for 2  h and then incubated with specific primary antibodies for 2  h or overnight at 4c . after being washed with tris - buffered saline with tween ( tbst ) thrice , membranes were incubated with peroxidase - conjugated secondary antibodies for 1  h. finally , membranes were washed with tbst and developed by enhanced chemiluminescence . blots were quantified using amersham imager 600 and analyzed with image j. total rna was purified using trizol reagent and converted into cdna by superscript iii reverse transcriptase . the sequences of primers for pcr amplification were as follows : gapdh : forward , 5-aggtcggtgtgaacggatttg-3 ; reverse , 5-tgtagaccatgtagttgaggtca-3 ; ppar- : forward , 5-ttttcaagggtgccagtttc-3 ; reverse , 5-tctgtgacgatctgcctgag-3 ; fabp4 : forward , 5-tccagtgaaaactttgatgattat-3 ; reverse , 5-acgcattccaccaccagtttatca-3 . quantitative pcr analysis was performed using sybr select master mix in 7500 fast real - time pcr system . the thermal profile for real - time pcr was 50c for 2  min , 95c for 10  min , followed by 40 cycles of 95c for 15  s and 60c for 1  min , and then 95c for 30  s and 60c for 15  s. relative expression was calculated using the 2 method . the level of peripheral blood hmgb1 was detected in mice at 16 weeks in accordance with the elisa kit 's protocol . the spectrophotometry ( od ) value of the microplate reader was set at 450  nm wave length . one - way analysis of variance with bonferroni 's corrections was used for multiple comparisons . to determine the changes in hmgb1 and autophagy protein lc3 in epididymal adipose tissues in obese mice , male c57bl/6 mice were fed with hfd for 16 weeks . the mean body weight for each group is shown in figure 1(a ) . with the extension of time , after 16 weeks , the mean body weight of the hfd group was about 1.5 times that of the nd group . moreover , the epididymal adipose tissue of the hfd group increased notably ( figures 1(b ) and 1(c ) ) . we also found that the adipocyte sizes in the hfd group were significantly larger than those in the nd group ( figure 1(d ) ) . hmgb1 was assessed in epididymal adipose tissue and serum by western blot and elisa , respectively . compared with the nd group , the hfd group showed that hmgb1 expression increased in both epididymal adipose tissues and peripheral blood of obese mice ( figures 1(e ) , 1(f ) , and 1(h ) ) . western blot was also used to detect the expression of autophagic protein lc3 in epididymal adipose tissues . the lc3ii protein markedly increased , reaching 3.29 times that of the normal - diet groups ( figures 1(f ) and 1(g ) ) . these data suggested that hfd promoted hmgb1 production and activated autophagy in the epididymal adipose tissues in mice . to determine the functional role of exogenous hmgb1 in autophagy , 3t3-l1 preadipocytes were treated with 0.2  g / ml recombinant hmgb1 protein for 24  h , which did not affect the normal cell proliferation   and hmgb1 protein expression in cells ( figures 2(a ) and 2(d ) ) . however , compared with the control group , the treatment group showed that the expression of autophagic protein lc3ii increased ( figures 2(a ) and 2(b ) ) , whereas the p62 protein expression decreased significantly ( figures 2(a ) and 2(c ) ) . western blot analysis revealed that the knockdown of hmgb1 significantly reduced hmgb1 protein expression ( figures 3(a ) and 3(b ) ) . furthermore , the lc3ii protein decreased obviously , whereas the p62 protein increased markedly compared with the control group ( figures 3(c ) and 3(d ) ) . to further explore the effects of hmgb1 on autophagy in various differentiation periods of 3t3-l1 adipocytes , we added 0.2  g / ml hmgb1 protein in the differentiation stages of adipocytes for 24  h. oil - red - o staining was performed to detect intracellular lipid droplets . as shown in figures 4(a ) and 4(b ) , 3t3-l1 preadipocytes were successfully induced into the adipocytes in the control group . however , after adding exogenous hmgb1 for 24  h , the lc3ii expression increased in the differentiation on the second and fourth days but decreased in the differentiation on the eighth and tenth days compared with the control group ( figures 4(c ) and 4(e ) ) . on the contrary , the p62 protein expression decreased in the differentiation on the second and fourth days ( figures 4(c ) and 4(f ) ) . finally , the expression of adipocyte differentiation related genes peroxisome proliferator - activated receptor- ( ppar )  and fabp ( fatty acid - binding protein ) 4 was measured . no significant difference in ppar- was found between the two groups ( figure 4(g ) ) , but the fabp4 expression significantly decreased in 4 days and increased in 8 days ( figure 4(h ) ) . these data indicated that the hmgb1 protein exerted different effects on autophagy and lipid metabolism in the early and late stages of adipocyte differentiation . white adipose tissue is primarily responsible for energy storage of the body , whereas brown adipose tissue is mainly responsible for energy degradation through uncoupling protein 1 ( ucp1 ) . obesity is characterized by an excess of white adipose tissue   and leads to adipocyte dysfunction , thereby increasing the risk for insulin resistance , type 2 diabetes mellitus , and cardiovascular diseases [ 26 , 27 ] . hmgb1 is produced as a damage - associated molecular pattern molecule ( damp ) by necrotic cells or activated immune cells . our data also confirmed the increased expression of hmgb1 in the adipose tissues and peripheral blood of mice induced by hfd . autophagy is a process which passes intracytoplasmic ingredients to lysosomes for degradation and provides energy to the body . autophagy not only is very important in the maintenance of cellular homeostasis but also participates in the metabolism of lipid droplets and lipogenesis . research showed that , in both in vivo and in vitro experiments , the inhibition of autophagy increases lipid storage . in addition , knockdown of autophagy genes atg5 and atg7 in adipocytes can improve insulin sensitivity and relieve obesity . the suppression of autophagy also reduced the expression of adipogenesis - related genes , such as ppar- and c / ebps . previous studies proved that the knockdown of atg7 decreased tg accumulation in 3t3-l1 preadipocytes , generated obese mice with decreased weight and white adipose mass , and then enhanced insulin sensitivity . some research found that hmgb1 activated an autophagic response to oxidative stress , and the loss of hmgb1 inhibited autophagy . we observed the change of autophagy in 3t3-l1 preadipocytes when hmgb1 was disturbed by specific small interfering rna . our results showed that autophagy was weakened when the expression of hmgb1 was suppressed in 3t3-l1 preadipocytes . hmgb1 also played a vital role in regulating autophagy in response to metabolic stress and oxidative damage . however , little is known about the connections between the hmgb1 protein and the change of autophagy in adipose tissues of mice . our results displayed that hmgb1 increased along with autophagy activated in adipose tissue of obese mice induced by hfd . this finding implied that activated autophagy may be a protective mechanism under cell stress state induced by hfd . to gain insights into the effects of hmgb1 on autophagy in adipocytes , in vitro studies were performed in adipocytes . 3t3-l1 preadipocytes were first stimulated with the recombinant hmgb1 protein to observe the expression of autophagy - related protein lc3 ( microtubule - associated protein 1a/1b - light chain 3 ) and p62 . a cytosolic form of lc3 ( lc3i ) is conjugated to phosphatidylethanolamine to form lc3-phosphatidylethanolamine conjugate ( lc3ii ) , which is recruited to autophagosomal membranes [ 34 , 35 ] . thus , the formation of lc3ii appears to be correlated with the induction of autophagy . p62 serves as a link between lc3 and ubiquitinated substrates , and the inhibition of autophagy correlates with the increased levels of p62 . our results showed that lc3ii increased and p62 decreased when cells were treated with recombinant hmgb1 protein . this finding suggests that exogenous hmgb1 induced autophagy in 3t3-l1 preadipocytes . in addition , as small interfering rna knocked down the expression of hmgb1 , autophagy appeared to be weakened . these data indicated that hmgb1 played an important role in regulating the autophagy in 3t3-l1 preadipocytes . we observed hmgb1 activated autophagy in the early differentiation within 2 and 4 days but weakened autophagy in differentiation after 8 and 10 days . presumably , the activation of autophagy resisted external stress in the early differentiation stage but was unable to resist this constant pressure in the late stage of differentiation . these results demonstrated that hmgb1 played a different role in the differentiation of 3t3-l1 adipocytes . several transcription factors , such as ppar- , are involved in the differentiation of preadipocytes into mature adipocytes . fabp4 , a lipid chaperone , is expressed in adipocytes and plays an important role in the regulation of insulin sensitivity . this finding was consistent with the expression of lc3 , which suggested that hmgb1 regulated autophagy and further altered lipid metabolism in adipocytes . these results not only provided a new theoretical basis for hmgb1 and autophagy in adipocytes but also presented a new target for the treatment of obesity and its related metabolic diseases .