obesity is the most common metabolic disorder and results from the combined effects of excess energy intake and reduced energy expenditure .
obesity is associated with a variety of clinical disorders , including hypertension , insulin resistance , glucose intolerance , and dyslipidemia .
it is well known that an oversupply of fat is associated with the development of obesity in mice .
long - term feeding with a high - fat diet can induce obesity with hyperlipidemia , insulin resistance , hyperphagia , and hypergluconemia [ 4 , 5 ] .
nonalcoholic fatty liver disease ( nafld ) is present in up to one - third of the general population and affects all ages and ethnic groups .
nafld is the second leading cause of death in the general population [ 6 , 7 ] . at present
, there is no pharmacological agent known to reverse nafld and effective medical interventions have focused on the modification of risk factors , such as weight reduction and diet .
mistletoe is a hemiparasitic plant growing all over the world on various deciduous trees , such as oak .
korean mistletoe ( km , viscum album cololatum ) extract ( kme ) has been shown to have anticancer , antioxidant , antidiabetes   and antidementia   effects , and it enhances immune system function .
cellular power plants  because they generate most of the cellular supply of adenosine triphosphate ( atp ) which is used as a source of chemical energy .
studies have implicated mitochondria in several human diseases , including metabolic diseases , cardiac dysfunction , mental disorders , and the aging process .
decreased mitochondrial respiration rates   and reduced expression of genes involved in mitochondrial oxidative capacity   have been reported in diet - induced obese rats . because kme improves mitochondrial function , we asked whether it has beneficial effects on obesity .
it is well known that adipocyte differentiation and the extent of subsequent fat accumulation are closely related to the occurrence and advancement of various diseases , such as coronary artery disease and obesity [ 1 , 22 , 23 ] .
3t3-l1 cells have served as a useful in vitro model for adipocyte differentiation and function .
the differentiation of preadipocytes into adipocytes requires a variety of effectors that activate a cascade of transcription factors , such as ccaat / enhancer - binding protein- ( c / ebp- ) , peroxisome proliferator - activated receptor ( ppar- ) , and sterol regulatory element binding element protein-1c ( srebp-1c ) .
this cascade begins with the ccaat enhancer - binding protein- ( c / ebp- )  and  , which induces the expression of c / ebp- and ppar- [ 2527 ] .
these transcription factors coordinate the expression of genes involved in creating and maintaining the adipocyte phenotype , including genes for adipocyte fatty acid - binding protein , glucose transporter 4 , lipoprotein lipase ( lpl ) and leptin [ 28 , 29 ] .
activated srebp-1c accelerates adipogenesis through the overexpression of adipogenic enzymes , such as fatty acid synthase(fas ) , acyl - coa synthase ( acc ) , and acyl - coa carboxylase ( acs ) .
lpl is the major enzyme that hydrolyzes triglyceride ( tg ) molecules of chylomicrons and vldl particles .
the released free fatty acids are either oxidized to generate atp in muscle or stored in adipose tissue .
we measured the inhibitory effect of kme on adipogenic factors in 3t3-l1 cells and on the development of obesity and nafld in mice fed a high - fat diet .
our data show that kme suppresses adipocyte differentiation through down - regulation of adipogenic factors and could ameliorate obesity and nafld in mice fed a high - fat diet .
our results indicate the great potential of kme as a potential metabolic regulator of adipocyte differentiation and a potential therapeutic agent for preventing or treating obesity and nafld .
mistletoe growing on oak was harvested from gangwon - do , republic of korea , in february .
the mistletoe was 1 or 2 years old , and the leaves , stems , and fruits were cut into 2 joints from the end of a branch , washed with distilled water ( dw ) , and dried .
. then mistletoe leaves , fruits , and stems were freeze - dried , crushed and ground in approximately 10 volumes of dw for 30 seconds . after being washed ,
they were ground in a mixer for 2 minutes and stirred for 16 hours at 4c . to obtain fine mistletoe extract ,
the homogenized mistletoe was centrifuged at 8,000  rpm for 30 minutes at 4c , and the resulting supernatant was successively filtered through different pore sizes ( 0.9 and 0.45  m ) .
the mistletoe extract was freeze - dried and resuspended in dw at an appropriate dilution factor .
lean , male c57bl/6 mice ( 7 weeks old ) were purchased from central lab animal inc .
all mice were housed for 1 week under a 12/12-hour light / dark cycle in a temperature(22  1c ) and humidity(55  5% ) controlled room and fed standard laboratory chow and water ad libitum .
kme was mixed with either powdered chow ( m07 , feedlab , republic of korea ) or high - fat ( d12327 , research diets , inc .
, new brunswick , nj , usa ) feed at a concentration of 4  g / kg of food to provide a 3000  mg / kg / day ( mpk ) dose .
these dietary amounts represent the maximum amount of chow that these animals were able to consume during a 24-hour period .
seven c57bl/6 mice on the above ad libitum diets had their daily average caloric intake and weekly body weight measurement during the course of the study . at the conclusion of the in vivo experiment ,
the mice were sacrificed and the epididymal pads were collected and weighed . the adaptive thermogenic response was measured in a cold test , during which the animals were individually housed at 4c for 6 hours .
liver tissues were isolated immediately after sacrifice . for hematoxylin and eosin ( he ) staining ,
tissues were fixed in 10% formalin and processed and embedded in paraffin prior to sectioning ( 10  m . ) and staining .
liver biopsies for electron microscopy were cut into 1  mm pieces , fixed immediately after collection in karnovsky fixative ( glutaraldehyde in cacodylate buffer ) , and kept at 4c . the second step involved postfixation with 1% osmium tetraoxide in 0.1  m cacodylate buffer for 1 hour at 4c .
tissues were then dehydrated through successive baths of graded alcohol , followed by a propylene oxide bath and a treatment with propylene oxide and resin mix before being embedded in a pure epoxy resin ( araldite , epon 812 ) that solidified after 48  h at 60c .
semithin sections were cut at 2  m , stained with toluidine blue , and then analyzed by light microscopy .
ultrathin sections were cut at 70  nm and examined with an electron microscope ( bx 50 , olympus , japan ) .
electron micrographs ( 400x magnification ) were digitized and analyzed using acd see , version 4.0 .
the liver of 1 mouse from each group ( control , high - fat diet ( hfd ) , hfd + kme 3000  mg / kg ) was measured . in brief , the following criteria were used for scoring hepatic steatosis : grade 0 , grade 1 , hepatocytes occupying < 33% of the hepatic parenchyma ; grade 2 , fatty hepatocytes occupying 3366% of the hepatic parenchyma ; and grade 3 , fatty hepatocytes occupying > 66% of the hepatic parenchyma .
mice were subjected to an endurance test using a variable - speed belt treadmill enclosed in a plexiglass chamber with a stimulus device consisting of a shock grid attached to the rear of the belt ( columbus instruments oxymax system , columbus , oh , usa ) .
the shock grid was set to deliver 0.2  ma , which caused an uncomfortable shock but did not physically harm or injure the animals .
mice were run at 16.2 meter / minutes for 10 minutes with a 5 incline . for high - fat- ( hf- ) fed animals ,
the speed was gradually increased from 10.8 to 24.6 meter / minutes and then maintained until exhaustion .
exhaustion was determined to have been reached if the mice were unable to run for 10 seconds , at which point the electric shock was discontinued .  
3t3-l1 preadipocytes purchased from atcc ( american type culture collection , manassas , va , usa ) were cultured in dulbecco's - modified eagle 's medium ( dmem , hyclone , logan , ut , usa ) containing 10% bovine calf serum ( bcs , hyclone , logan , utah , usa ) at 37c in a humidified atmosphere of 5% co2 . after 3 or 4 days , the cells had reached 90% confluence and were collected with 0.05% trypsin/0.53  mm edta treatment .
after centrifugation ( 1300  rpm , 5 minutes ) , the cells were plated in 6-well plates at a concentration of 3  10 cell / well .
one day after confluence ( designated  day 0  ) , cell differentiation was induced with a mixture of methylisobutylxanthine ( 0.5  mm ) , dexamethasone ( 0.25  mm ) , and insulin ( 5  mg / ml ) in dmem containing 10% fetal bovine serum ( fbs ) . on day 2 and thereafter , dmem supplemented with 10% fbs and 5  mg / ml insulin was replaced every 2 days .
3t3-l1 adipocytes , 7 to 8 days after differentiation , were treated with kme 6  g/l or phosphate - buffered saline ( pbs ) for 24 hours .
total rna was isolated from 3t3-l1 adipocytes using trizol reagent ( invitrogen , carlsbad , ca , usa ) .
cells were homogenized by 4 - 5 passages through a 23-gauge needle . after a 5-minute reaction , samples were mixed with chloroform by vortex machine for 15 seconds .
total rna was precipitated by mixing with isopropyl alcohol and centrifuging at 12,000  rpm .
the resulting rna pellets were washed with 75% ethanol and dissolved in rnase - free water .
total extracted rna ( 1  g ) mixed with annealing oligo dt primer and rnase - free water was incubated in a thermocycler for denaturing rna .
reactant was mixed with 5x first strand buffer , 20  mm dtt , 10  mm dntp mix , rnase - free water , and superscript ii reverse transcriptase .
the mixtures were incubated in a thermocycler ( 42c for 1 hour and 72c for 7 minutes ) to generate cdna .
real - time rt - pcr analysis was performed with an ab 7500 real - time pcr system ( applied biosystems , foster city , ca , usa ) .
samples containing 2x sybr green pcr master mix ( applied biosystems , foster city , ca , usa ) , 0.5 mol each of appropriate primers , and cdna were incubated in the ab 7500 real - time pcr system for an initial denaturation at 94c for 10 minutes , followed by 40 pcr cycles . each cycle
-actin was used as an internal housekeeping control . to confirm amplification of specific transcripts ,
melting curve profiles were produced at the end of each pcr cycle by cooling the sample to 65c for 15 seconds and heating slowly to 95c , with continuous fluorescence measurement .
data ( means  se ) were analyzed using graphpad prism ( version 5.04 , graphpad software , usa ) .
unpaired two - tailed student 's t - tests were used to evaluate differences between means , as indicated .
during the 15-week experiment , body weight and food intake were measured weekly . as shown in figure 1 ,
the kme - treated group had a slight decrease in body weight compared to the control group .
however , this effect was significantly pronounced in animals that were fed an hf diet with kme .
the difference between the kme - treated and control animals became significant at week 9 . at the end of the experiment ,
the body weight of mice fed kme was 20% lower ( p < 0.05 ) than that of the control group , whereas hf diet plus kme mice weighed almost the same as the mice that ate normal chow ( figure 1(a ) ) .
these effects of kme on body weight were not due to decreased food intake , as the amount of kcal consumed per mouse over a 24-hour period remained unchanged ( figure 1(b ) ) .
the data indicate that kme might have antiobesity effects in vivo , without affecting food intake . to test whether body weight loss was caused by decreased adiposity
this analysis revealed that epididymal white adipose tissue was significantly reduced in the hf diet plus kme group ( figure 1(c ) ) .
we performed a cold test to assess the effect of kme on adaptive thermogenesis capacity .
kme - treated animals maintained higher body temperatures than nontreated animals ( figure 2(a ) ) , suggesting that it improved this capacity . to test whether thermogenesis is accompanied by changes in the expression of genes involved in thermogenesis ,
we examined mrna levels of ucp1 , the major contributor to heat production , by real - time rt - pcr .  
figure 2(b ) shows that the mrna level of ucp1 was remarkably increased in kme - treated mice .
because kme reduced body weight ( figure 1(a ) ) and increased thermogenesis ( figure 3 ) , we evaluated the effect of kme administration on a treadmill endurance test .
the mice were trained to run at 16.2 meter / minutes for 10 minutes at a 5 incline the day before the running test , according to the procedure used for our previous report .
the experiment was initiated at 10 meter / minutes at a 0 incline with a gradual increase in speed .
the mice were run until exhaustion , which was defined as remaining on the shock grid for longer than 10 consecutive seconds .
surprisingly , kme - treated mice ran twice as far as high - fat diet mice ( figure 3 ) .  
one of the most common characteristics among people with obesity is the development of fatty liver [ 33 , 34 ] .
histologic evaluation is regarded as the  gold standard  for assessing the presence and severity of nafld .
we histologically evaluated liver sections to determine the extent to which kme attenuated hepatic steatosis development .
as shown in figure 4 , the control group exhibited little histologic evidence of hepatic steatosis .
however , a marked reduction in the degree of steatosis was seen in livers from high - fat diet mice treated with kme .
furthermore , hepatic steatosis scores were dramatically lower in the high - fat diet mice fed kme ( grade 1 ) . to demonstrate the inhibitory mechanism of kme on adipocyte differentiation
, we measured the expression levels of the transcription factors ppar- , c / ebp- , and srebp-1c in 3t3-l1 cells treated with 6  g/l of kme .
our results show that ppar- , c / ebp- , and srebp-1c mrna levels were decreased by 64% , 60% , and 32% , respectively , ( figure 5(a ) ) .
we also measured the mrna expression levels of adipogenic enzymes , such as fas , acs , and acc . we found that they were reduced by 69% , 55% , and 22% , respectively ( figure 5(b ) ) .
lpl expression is not directly associated with lipid synthesis but is strongly associated with adipogenesis .
therefore , we measured the expression level of lpl mrna and found that the level of expression in kme - treated 3t3-l1 cells was decreased by 89% compared to that of untreated cells ( figure 5(a ) ) .
our results showed that body weight gain in groups fed a diet supplemented with kme was reduced compared to control hfd mice .
this study also provides evidence that dietary supplementation of kme protects against hepatic steatosis development .
we have considered the possibility that the effect of kme may be mediated by food intake because decreased food intake would be expected to significantly affect body weight , which influences hepatic steatosis . in this study , however , food intake did not differ between the groups .
this suggests that kme directly protected against obesity and hepatic steatosis independent of food intake .  
brown adipose tissues ( bats ) use stored triacylglycerols ( tg ) to maintain body temperature . these cells produce energy from fatty acid metabolism to generate heat through the action of uncoupling protein 1 ( ucp1 ) , a mitochondrial protein found only in brown adipose tissue .
brown adipocytes contain less tg and more mitochondria than white adipocytes , resulting in their unique color .
kme treatment increased ucp1 expression levels in bat , which poised the mitochondria for respiration uncoupling .
this effect is consistent with the observed enhancement of cold tolerance and helps explain the increase in energy expenditure and resistance to weight gain .  
this is most evident in muscle , a metabolically flexible tissue that switches between lipid and carbohydrate substrates to fulfill energy requirements .
one study found that mitochondrial oxphos activity within the oxidative soleus muscle , which is resistant to fatigue and is dependent on mitochondrial activity for atp production , was more affected than in the glycolytic muscle tibialis anterior .
the main changes included a reduction in respiratory chain activity with a concomitant decrease in mitochondrial atp production . in line with this finding and previously ,
kme significantly improved muscle oxidative capacity , resulting in enhancing the endurance . to further consolidate our hypothesis that kme inhibits obesity
, we evaluated the effects of kme treatment in vitro using an adipocyte differentiation development system , which is regulated by transcriptional activators such as ppar- , c / ebp- , and some transcriptional regulators at the molecular level [ 39 , 40 ] .
ppar- is a member of the nuclear receptor superfamily of transcription factors and is dominantly expressed in adipose tissue .
srebp1 is known to critically cross - activate a ligand binding domain of ppar- and activate the production of an endogenous ppar- ligand .
adipogenesis can be induced through the action of some enzymes , such as fas , acc , and acyl - coa synthetase ( acs ) .
the expressions of these genes are regulated by transcription factors , including ppar- , c / ebp- , and srebp1 , which are known to be crucial activators for adipogenesis and showed early changes in gene expression during adipocyte differentiation [ 44 , 45 ] . in this study , we showed that kme treatment significantly decreased srebp-1c , c / ebp- , and ppar- mrna expression in cultured 3t3-l1 adipocytes and inhibited expression of adipocyte - specific proteins ( fas , acc , acs , and lpl ) . collectively , these observations suggest that kme suppresses adipocyte differentiation by regulating the expression levels of adipogenic transcription factors .
further investigation is necessary to define specific mechanisms by which kme protects against obesity - mediated hepatic steatosis . to elucidate the mechanism associated with the antiobesity effects of kme
kme had marked inhibitory effects on the development of obesity and nafld in mice fed high - fat diets . activating endurance capacity and increasing thermogenesis are two possible mechanisms for the antiobesity effect of kme .
this finding suggests that kme may be a potential dietary strategy for preventing obesity and nafld .
 this study investigates the inhibitory effects of korean mistletoe extract ( kme ) on adipogenic factors in 3t3-l1 cells and obesity and nonalcoholic fatty liver disease ( nafld ) in mice fed a high - fat diet . 
 male c57bl/6 mice fed a high - fat diet were treated with kme ( 3  g / kg / day ) for 15 weeks for the antiobesity and nafld experiments . 
 body weight and daily food intake were measured regularly during the experimental period . 
 the epididymal pad was measured and liver histology was observed . 
 the effects of kme on thermogenesis and endurance capacity were measured . 
 the effects of kme on adipogenic factors were examined in 3t3-l1 cells . 
 body and epididymal fat pad weights were reduced in kme - treated mice , and histological examination showed an amelioration of fatty liver in kme - treated mice , without an effect on food consumption . 
 kme potently induces mitochondrial activity by activating thermogenesis and improving endurance capacity . 
 kme also inhibited adipogenic factors in vitro . 
 these results demonstrate the inhibitory effects of kme on obesity and nafld in mice fed a high - fat diet . 
 the effects appear to be mediated through an enhanced mitochondrial activity . 
 therefore , kme may be an effective therapeutic candidate for treating obesity and fatty liver caused by a high - fat diet . 