increasing evidence suggests that inflammation plays an important role in the development of insulin resistance .
the inflammatory status of the liver and adipose tissue , the main insulin - resistance - related organs , is critical for insulin sensitivity of the whole body [ 3 , 4 ] . during development of inflammation in the liver
, hepatocytes are exposed to the increasing cytokines such as tumor necrosis factor- ( tnf- ) , interleukin-6 ( il-6 ) , and monocyte chemoattractant protein-1 ( mcp-1 ) .
these cytokines were regulated by nuclear factor kappa - b ( nf-b ) [ 6 , 7 ] .
a number of studies demonstrated that hepatic nf-b activation was linked to the development of the whole body inflammation , resulting in insulin resistance [ 8 , 9 ] .
additionally , adipose tissue is an active endocrine organ that secretes numerous inflammation - related adipokines , cytokines , and chemokines .
the expression of tnf- , il-6 , mcp-1 , and leptin is upregulated in the adipose tissue of obese mice which is closely associated with insulin resistance [ 1012 ] .  
interestingly , some studies reported that oxidative stress could induce inflammation during development of insulin resistance [ 13 , 14 ] .
oxidative stress which is induced by excess reactive oxygen species ( ros ) could activate nf-b .
antioxidants have been reported to potently suppress the activation of nf-b in the liver [ 1618 ] .
furthermore , increased oxidative stress in adipocytes causes dysregulation of cytokines and adipokines , including adiponectin , mcp-1 , il-6 , and plasminogen activator inhibitor-1 ( pai-1 ) [ 19 , 20 ] .
accordingly , antioxidants have been also reported to decrease inflammation in adipose tissue and ultimately ameliorate insulin resistance [ 21 , 22 ] .
apocynin ( 4-hydroxy-3-methoxy - acetophenone ) is a constituent of the himalayan herb picrorhiza kurrooa royle ( scrophulariaceae ) which is regarded as an inhibitor of nicotinamide adenine dinucleotide phosphate ( nadph)-oxidase and is widely used as an antioxidant in research .
in addition , apocynin also exhibits anti - inflammatory effects in previous studies [ 23 , 24 ] .
tnf- protein expression in coronary arteries , decreased vascular cell adhesion molecule-1 ( vcam-1 ) induced by tnf- in vein endothelial cells , reduced polymorphonuclear granulocyte chemotaxis , inhibited peroxynitrite in airway lumen in mice , attenuated inflammation - mediated cartilage destruction activity in human articular cartilage , and reduced cyclooxygenase ( cox)-2 expression in human monocytes . since the critical role of inflammation in genesis of insulin resistance , it is reasonable to speculate that apocynin could improve insulin resistance through suppressing inflammation .
however , no studies were conducted to examine the effects of apocynin on inflammation and its subsequent effects on insulin resistance . in this study
, c57bl/6 mice were fed with high - fat diet ( hfd ) to establish insulin resistance for investigating the effects of apocynin on the expression of inflammatory factors in blood , liver , and adipose tissue , and then evaluating the improvement of insulin sensitivity with apocynin treatment .
four - week - old male c57bl/6 mice used in this study were purchased from animal center , yangzhou university ( yangzhou , jiangsu , china ) .
the mice were fed either with high - fat diet ( 60% kcal fat , 20% kcal carbohydrates , 20% kcal protein , guangzhou animal experiment center , guangzhou , china ) , or with normal chow diet ( 10% kcal fat , 70% kcal carbohydrates , 20% kcal protein ) after one week 's acclimation period .
mice were kept on a 12-hour light , 12-hour dark cycle and fed ad libitum .
after 12 weeks of feeding , the hfd - fed mice showed obvious phenotypes of insulin resistance compared to the normal diet control group ( nd - con ) .
one group received normal water ( hfd control group , hfd - con ) , while the other group received apocynin ( 2.4 
g / l ) dissolved in drink water ( apocynin group , hfd - apo ) for another five weeks with hfd .
after given ketamine - xylazine ( 130/8.8  mg / kg ) , their tissues were harvested , weighed , snap frozen in liquid nitrogen , and stored at 80c until use . dietary intake and body weight were recorded every week .
mice were fasted for 12 hours , and blood samples were collected from the orbital sinus under anesthesia .
after centrifugation at 15,000  g for 1  min , the supernatants of the blood samples were separated and subjected to measurements of leptin , tnf- , and il-6 levels with the elisa kits ( raybiotech , norcross , ga , usa ) .
plasma insulin was determined with mouse insulin elisa assay kit ( linco , charles , mo , usa ) .
glucose was injected intraperitoneally with the concentration of 1  g / kg body weight .
blood glucose measurement were obtained from the tail at 0 , 30 , 60 , and 120  min .
blood glucose levels were determined at indicated intervals with one touch profile glucose meter ( johnson & johnson , new brunswick , nj , usa ) .
total rna was extracted from the liver and epididymal adipose tissue of mice by trizol ( takara bio , shiga , japan ) .
synthesis of cdna was performed by m - mlv reverse transcriptase ( toyobo , osaka , japan ) .
quantitative real - time pcr was performed with sybr premix ex taq ( takara bio , shiga , japan ) , using the stepone real - time pcr system according to the manufacturer 's instructions ( applied biosystems , foster city , ca , usa ) .
nuclear protein extracts were prepared from liver tissue with the nuclear extract kit ( active motif , carlsbad , ca ) according to the manufacturer 's instructions .
protein concentrations were then quantified with a bradford assay , and equal amounts of protein were used in a colorimetric nf-b assay specific for the activated form of p65 subunit of nf-b ( trans am nf-b p65 kit ; active motif , carlsbad , ca ) .
p values were determined by one - way anova followed by lsd and differences were considered significant if p < .05 .
the effect of apocynin on body weight in hfd - fed mice was shown in figure 1(a ) .
the body weight of hfd - apo group and hfd - con group were similar ( p > .05 ) and significantly higher than that in the nd - con group until 3 weeks after the treatment of apocynin ( p < .05 ) . from the fourth week to the end of the experiment ,
the body weight of hfd - apo group was clearly lower than that of hfd - con group ( p < .05 ) .
the mean dietary intake during the experiment was significantly lower in the hfd - con and hfd - apo groups than that in the nd - con group ( p < .05 , figure 1(b ) ) , while energy intake has no significant difference among groups ( p > .05 , figure 1(c ) ) .
ipgtts were performed before the sacrifice of the mice to assess glucose tolerance ( figure 2(a ) ) .
area under the curve ( auc ) parameter was employed as the index of glucose tolerance ( figure 2(b ) ) .
auc was significantly higher in the hfd - con group compared to the nd - con group ( p < .05 ) .
the hfd - apo group had remarkably lower auc than the hfd - con group ( p < .05 ) . at the end of the ipgtts ,
fasting glucose and fasting insulin levels were significantly higher in hfd - con group than nd - con group ( p < .05 ) .
apocynin administration significantly improved fasting glucose ( figure 2(c ) ) and fasting insulin in hfd - apo group compared to the hfd - con group ( figure 2(d ) ) ( p < .05 , resp . ) serum inflammatory cytokines levels are shown in figures 3(a)3(c ) .
the levels of tnf- , il-6 , and leptin of hfd - con group were remarkably higher than nd - con group ( p < .05 , resp . ) .
apocynin treatment significantly decreased the levels of tnf- , il-6 , and leptin in the serum ( p < .05 , resp . ) .
in adipose tissue , the hfd - con group exhibited significantly higher gene expression of tnf- , il-6 , mcp-1 , and leptin and lower expression of adiponectin than nd - con group ( p < .05 , resp . ) .
apocynin treatment obviously decreased tnf- , il-6 , mcp-1 , and leptin expression ( p < .05 , resp . ) ( figures 4(a)4(d ) ) .
however , there was no change in expression of adiponectin after treatment with apocynin ( p > .05 ) ( figure 4(e ) ) . in the liver ,
gene expression tnf- , il-6 , and mcp-1 were significantly lower in the hfd - apo than the hfd - con group ( p < .05 , resp . ) ( figures 5(a)5(c ) ) .
we further tested nf-b activity in liver nuclear extract samples from the mice of three experiment groups .
we found that significantly less nf-b activity was derived from apocynin - treated mice than those derived from untreated hfd - fed mice ( p < .05 ) ( figure 5(d ) ) .
our present study indicates that apocynin administration in hfd - fed c57bl/6 mice can improve insulin sensitivity , reduce the diverse plasma inflammatory cytokines , suppress gene expression of inflammation - related molecules in both liver and adipose tissue , and decrease the activity of transcription factor nf-b in liver tissue .
these results suggest that apocynin could ameliorate insulin resistance through the suppression of inflammation in hfd - fed c57bl/6 mice . to reflect modern dietary conditions , we employed c57bl/6 mice fed with hfd ( 60% kcal from fat ) for 12 weeks which is a simple insulin resistance model .
hfd - fed mice exhibited higher body weight than normal diet mice . although food and energy intakes were not significantly different , the body weight of the hfd - con group was increased , whereas that of the hfd - apo group was decreased and close to nd - con group .
the hfd - fed mice exhibited significantly higher fasting glucose , fasting insulin levels , and auc of ipgtt than normal control mice .
recently , there have been a few studies that implicated that apocynin may improve insulin resistance which are consistent with the results of the present study .
winiarska and colleagues found that apocynin could inhibit renal gluconeogenesis action by suppressing oxidative stress , resulting in hypoglycaemic response in diabetic rabbits .
another study indicated that apocynin administration decreased lipid peroxidation and hydrogen peroxide in adipose tissue which led to the improvement of plasma glucose and insulin resistance in kkay mice , a transgenic animal model of type 2 diabetes . also , the antioxidative effect of apocynin was protective in diabetic nephropathy [ 34 , 35 ] , in diabetic endothelial dysfunction [ 36 , 37 ] , and in diabetic mitochondrial dysfunction in skeletal muscle .
however , none of the previous studies focused on the anti - inflammatory effect of apocynin on insulin resistance .  
in contrast , suppressing inflammatory factors may ameliorate insulin resistance [ 39 , 40 ] .
the molecular pathways that link inflammation and insulin resistance include a variety of cytokines and adipocytokines such as tnf- , il-6 , mcp-1 , leptin , and adiponectin .
high circulating levels of tnf- , il-6 , and leptin are recognized as markers of insulin resistance [ 10 , 41 , 42 ] .
the inflammatory status of the liver and adipose tissue could directly impact the systemic inflammatory status that accompanies insulin resistance .
inflammation - related transcription factor nf-b in the liver is regarded as an important factor for inducing insulin resistance . increased hepatic nf-b activity could cause profound hepatic insulin resistance and moderate systemic insulin resistance .
furthermore , as a transcription factor , nf-b regulates expression of many kinds of cytokines in the liver .
once nf-b is activated , the gene expression of tnf- , il-6 , and mcp-1 is inordinately increased [ 6 , 7 ] .
the endocrine function of adipose tissue and the association of obesity result in chronic low - grade inflammation and reinforce the concept of the central role of adipose tissue in mediating obesity - linked insulin resistance .
it has been discovered that the adipose tissue in fact contributes 1535% of the body 's basal circulating il-6 and a large rate of whole body tnf- .
adipose tissue macrophages ( atms ) are proved to be predominantly responsible for the elevated production of tnf-   and il-6 during obesity .
mcp-1 could contribute macrophage infiltration into adipose tissue and affect the number of atms in adipose tissue , which in turn impacts the expression of tnf- and il-6 .
so gene expression of leptin in adipose tissue directly affects leptin level in the blood .  
however , hart and his colleagues found that apocynin could reduce inflammation as well as superoxide anion .
afterwards , a number of studies have demonstrated that apocynin could modulate the production of inflammatory factors production both in vitro [ 2731 ] and in vivo [ 25 , 33 , 48 ] . in the present study , we investigated the effect of apocynin on the inflammatory status through hfd - induced insulin resistance in c57bl/6 mice .
our results show that the gene expression of tnf- , il-6 , and mcp-1 in the liver and the gene expression of tnf- , il-6 , mcp-1 , and leptin in adipose tissue were upregulated after hfd feeding in mice .
apocynin administration suppressed the expression of these inflammatory agents , resulting in the reduction of circulating tnf- , il-6 , and leptin levels that normally accompany insulin resistance .  
furthermore , we found that apocynin could reduce the activity of hepatic nf-b which contributes to the improvement of insulin resistance both in the liver and whole body .
there is compelling evidence that in many cell types , ros such as hydrogen peroxide and superoxide anion have a major effect on the release and nuclear translocation of the nf-b complex and on the induction of nf-b target genes [ 49 , 50 ] .
nadph oxidase , a major source of ros , triggers the production of superoxide anion [ 51 , 52 ] .
hence , it is reasonable that apocynin , an inhibitor of nadph oxidase , suppresses nf-b activity .
our results are in agreement with a study conducted by pechanova and his colleagues who used apocynin in spontaneously hypertensive rats .
after six weeks of treatment , apocynin significantly decreased the activity of nf-b in the left ventricle .
although in the present study , we have proved that apocynin could ameliorate inflammatory status and suppress the activity of nf-b in the liver , changes in other inflammatory signaling pathways and insulin signaling remain unknown .
apocynin has the potential to improve insulin sensitivity through ameliorating inflammatory status in the blood , liver , and adipose tissue of the c57bl/6 mice in which insulin resistance was induced by hfd .
 we investigated the effects of apocynin on high - fat diet- ( hfd- ) induced insulin resistance in c57bl/6 mice . 
 after 12 weeks of hfd , the mice that exhibited insulin resistance then received 5 weeks of apocynin ( 2.4  g / l , in water ) . 
 following apocynin treatment , fasting glucose , insulin , and glucose tolerance test showed significant improvement in insulin sensitivity in hfd - fed mice . 
 we demonstrated that serum levels of tumor necrosis factor- ( tnf- ) , interleukin-6 ( il-6 ) , and leptin were remarkably reduced with apocynin treatment . 
 we also found that mrna expression of tnf- , il-6 , and monocyte chemoattractant protein-1 ( mcp-1 ) in the liver and mrna expression of tnf- , il-6 , mcp-1 , and leptin in adipose tissue were suppressed by apocynin . 
 furthermore , the activity of transcription factor nf-b in the liver was significantly suppressed with apocynin treatment . 
 these results suggest that apocynin may reduce inflammatory factors in the blood , liver , and adipose tissue , resulting in amelioration of insulin resistance in hfd - fed mice . 
1. Introduction
2. Methods
3. Results
4. Discussion