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 .
 high mobility group box 1 protein ( hmgb1 ) is a molecule related to the development of inflammation . 
 autophagy is vital to maintain cellular homeostasis and protect against inflammation of adipocyte injury . 
 our recent work focused on the relationship of hmgb1 and autophagy in 3t3-l1 cells . in vivo experimental results showed that , compared with the normal - diet group , the high - fat diet mice displayed an increase in adipocyte size in the epididymal adipose tissues . 
 the expression levels of hmgb1 and lc3ii also increased in epididymal adipose tissues in high - fat diet group compared to the normal - diet mice . 
 the in vitro results indicated that hmgb1 protein treatment increased lc3ii formation in 3t3-l1 preadipocytes in contrast to that in the control group . 
 furthermore , lc3ii formation was inhibited through hmgb1 knockdown by sirna . 
 treatment with the hmgb1 protein enhanced lc3ii expression after 2 and 4 days but decreased the expression after 8 and 10 days among various differentiation stages of adipocytes . 
 by contrast , fabp4 expression decreased on the fourth day and increased on the eighth day . 
 hence , the hmgb1 protein modulated autophagy - related proteins and lipid - metabolism - related genes in adipocytes and could be a new target for treatment of obesity and related metabolic diseases . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusion