obesity abets several life style diseases such as diabetes type ii , atherosclerosis , or impaired wound healing . these adverse effects can be avoided or , if already present , reduced by physical activity .
exercise training and increased catecholamine levels affect adipocyte metabolism , on one hand by influencing insulin secretion   and on the other hand by additionally channelling the adipocyte metabolism towards lipolysis .
adenylyl cyclase transforms atp into cyclic amp ( camp ) , which in turn increases the activity level of camp - dependent protein kinase a ( pka ) [ 3 , 4 ] .
activation of the insulin receptor results in decreased lipolysis , increased lipogenesis and uptake of fatty acids .  
lipolysis is decreased by insulin - receptor - induced phosphodiesterase-3b ( pde-3b ) and 14 - 3 - 3beta - dependent reduction of pka activity [ 3 , 5 ] .
furthermore , the insulin receptor activates several lipogenic enzymes by tyrosine phosphorylation and promotes glut-4 recruitment to the cell membrane .  
protein tyrosine phosphatase interacting protein 51 ( ptpip51 ) is expressed in different organs with a multitude of functions [ 7 , 8 ] . as previously shown for mouse adipose tissue , ptpip51 acts as an effector in insulin signalling .
ptpip51 expression was correlated to the insulin resistance of the animals , according to the expression of its interaction partner protein tyrosine phosphatase 1b ( ptp1b ) .
in addition to that , ptpip51 is connected to the lipolytic mapk pathway by its interaction with raf-1 mediated by 14 - 3 - 3beta .
14 - 3 - 3beta also interacts with phosphodiesterase 3b , resulting in reduced intracellular camp levels and decreased pka activity . as 14 - 3 - 3beta and
ptp1b are important for adipocyte metabolism control , the role of its interaction partner ptpip51 needs to be disclosed .  
the interaction of ptpip51 with the insulin receptor and pka was investigated in adipose tissue from animals kept under different defined experimental conditions to search for a potential role of ptpip51 in adipocyte metabolism .
the experiments were performed with male c57bl/6 mice ( n = 40 ) and approved by the local animal care and use committee ( gi 20/24 nr .
the animals were kept under standard conditions ( 12  h light / dark cycle ) with no access to running wheels .
the experiments were run for 14 weeks , with a four - week period of fat feeding followed by a ten - week training period .  
control group ( sd ) : the animals were fed a standard diet ( altromin standard diet no . 1324 , altromin , lage , germany ) .  
endurance training control group ( sdt ) : the animals were fed a standard diet ( altromin standard diet no . 1324 , altromin , lage , germany ) and trained for a period of ten weeks .
the training was performed on a motorized treadmill for 35  min / day at 12  m / min , at 12% grade , 5 times per week , for ten weeks .
running speed was 0.27  m / s  0.05  m / s corresponding to 80% of maximal oxygen consumption .
high - fat - diet group ( hfd ) : the animals were fed a high - fat diet containing 45% fat for 14 weeks of the experimental period . high - fat - diet , training group ( hfdt ) : the animals were fed a high - fat - diet during the complete experimental time period . the endurance training period started four weeks after the beginning of high - fat - diet feeding and was similar to the training described above .
after 14 weeks , the animals were sacrificed in the morning between 10 and 12 o'clock and the abdominal adipose tissue was frozen in liquid nitrogen precooled isopentan , and transferred to 80c till further analysis .
a glucose tolerance test was performed to determine the insulin sensitivity of the mice [ 911 ] . for testing the glucose tolerance of the animals , they were injected intraperitoneally with 2 
g of 20% d - glucose dissolved in sterile 0.9% nacl solution per kg body weight .
the test was performed in the morning ; last feeding of the animals took place the previous day .
an antibody against a defined peptide sequence at the c - terminus of ptpip51 ( sequence : iqkdleelevilrd , exon 13 ) was produced ( biolux , stuttgart , germany ) .
identity and purity of the synthesized peptide were approved by esi - ms and uv - analysis .
the primary ptpip51 antibody to the c - terminus was used in 1  :  800 dilutions and visualized by alexa 555 ( molecular probes , darmstadt , germany , cat .
primary monoclonal mouse antibodies were used for double staining experiments with pka ( ab58187 , abcam plc , 330 cambridge science park , cambridge , uk ) and the beta - subunit of the ir ( clone ct-3 , millipore , 28820 single oak drive , temecula , ca , usa ) . to avoid unspecific binding of the primary mouse antibodies
, samples were preincubated with biotin - coupled anti - mouse - antibodies for one hour .
the reaction of the primary monoclonal mouse antibody was visualized using alexa fluor 488 secondary antibodies ( molecular probes , darmstadt , germany , cat .
confocal images of cells were obtained with a leica confocal laser scanning microscope ( clsm , 5 tcs sp2 , leica , bensheim , germany ) .
confocal images of alexa fluor 555 fluorescence were acquired using 6 plan - apochromat    40/1.4 oil objective , 548  nm excitation wavelengths ( helium - neon laser ) , and a 560585  nm band - pass filter .
the pinhole diameter was set to yield optical sections of 1 airy unit . for the detection of alexa fluor 488
, we used a plan - apochromat  40/1.4 oil objective , the 488  nm excitation wavelength of an argon laser , and a 505530  nm band - pass filter .
t3605 ) ( nuclear staining ) were acquired using plan - apochromat  40/1.4 oil objective , 633  nm excitation wavelengths ( helium - neon laser ) , and the 650670  nm bandpass filter .
the pinhole diameter was set to yield optical sections of 1 airy unit . acquired dic and confocal images
acquired images were subsequently processed by imagej ( v1.43  m ; rasband , w.s . , imagej , us national institutes of health , bethesda , md , usa , http://imagej.nih.gov/ij/ , 19972011 ) using an iterative deconvolution plug - in by bob dougherty ( http://www.optinav.com/imagej.html , iterative deconvolution ) . options were set for all confocal acquired images as follows : 8 numbers of iteration and 2.0  pixels of lp filter diameter .
point spread function was calculated for each channel separately by the imagej plug - in created by bob dougherty ( http://www.optinav.com/imagej.html , diffraction limit psf ) .
intensity correlation analysis ( ica ) was carried out using imagej ( v1.43  m ; rasband , w.s . ,
imagej , us national institutes of health , bethesda , md , usa , http://imagej.nih.gov/ij/ , 19972011 ) and an appropriate plug - in for ica included in the plug - in package of the wright cell imaging facility   ( http://www.uhnres.utoronto.ca/facilities/wcif/fdownload.html ) .
interaction of ptpip51 with either pka or beta - subunit of the ir was detected by the proximity ligation assay kit duolink ( olink |bioscience , uppsala , sweden , pla probe anti - rabbit minus for the detection of the rabbit ptpip51 antibody , cat .
90602 ; pla probe anti - mouse plus for the detection of the mouse anti pka or ir antibody , cat .
the dpla was performed according to the manufacturer 's protocol ; negative controls were included [ 9 , 14 , 15 ] . for the principle of the dpla , see figure 1 .
the axioplan 2 fluorescence microscope equipped with plan - apochromat objectives ( carl zeiss jena , germany ) was used for photo documentation . for visualization of the dpla ,
an excitation filter with a spectrum of 530560  nm and an emission filter with a spectrum of 572647  nm were used .  
the duolink image tool ( olink biosciences , uppsala , sweden ) was used for quantification of the detected dpla signals .
we accomplished a database research using the gps 2.1 database   to identify kinases within adipocyte metabolism that possess the ability to phosphorylate ptpip51 .  
the observed data were analysed by nonparametrical tests to determine the significance of the results .
the glucose tolerance was determined by a standard glucose tolerance test at the end of the experimental period .
sd and sdt animals displayed a slight increase of blood glucose levels 30 minutes after application of glucose which was almost normalized after 60 minutes .  
in hfd animals , blood glucose levels strongly increased from the basal level of 100  mg / dl to 400  mg / dl 60 minutes after application .
blood glucose levels did not return to normal values within 120 minutes . in hfdt animals ,
blood glucose levels increased to 270  mg / dl 30 minutes after application and almost returned to normal values after 120 minutes ( figure 2 ) .
the values of hfd , hfdt , and animals fed a standard diet ( sd and sdt ) are significantly different after 30 and 60 minutes .
after 120 minutes , the differences between standard diet and high fat diet animals remained significant , whereas there was no significant difference between standard diet and trained high - fat - diet animals .  
mean body weight was measured at different time points throughout the test ( see table 2 ) .  
statistical analysis showed that there was no significant difference between the four groups at 10 weeks of age . from the beginning of the training ,
the difference between standard diet and high - fat - diet - fed groups was highly significant , but there was no significant difference between the sd and sdt and , respectively hfd and hfdt groups .  
ptpip51 immunoreactivity was detected within the cytoplasm and at the plasma membrane ( figure 3 ) .  
the ir revealed a similar expression pattern when compared to ptpip51 . in the cytoplasm ,
co - localization is displayed in yellow to orange and non - co - localized parts are shown in blue .
the duolink proximity ligation assay ( dpla ) was performed to detect interactions between ptpip51 and the beta - subunit of the ir .
the interaction of ptpip51 and ir was verified as seen in figure 4 , where each dot indicates the interaction of both proteins .
this interaction of ptpip51 and the ir was found in the adipocytes of animals from all experimental groups .
the grade of interaction in adipose samples from sd animals corresponds to the level in hfdt animals .
the assumption of an interaction of ptpip51 and pka based on the database research was verified ( figure 4 ) .
the interaction levels of sd , hfd , and hfdt mice did not show significant differences .
to sum up the results , adipocytes from mice subjected to different diets and activity levels displayed a connection between ptpip51 and the insulin receptor , and ptpip51 and pka respectively .
the glucose - tolerance test revealed a glucose - intolerant status in hfd and hfdt animals , whereas the glucose tolerance of hfdt animals improved compared to hfd animals .  
immunofluorescence data displayed colocalization of ptpip51 with ir and pka suggesting an interaction between ptpip51 and ir , and ptpip51 and pka respectively .
search results displayed ptpip51 to be phosphorylated at tyrosine 176 by the insulin receptor and at serine 46 by pka .
these two phosphorylation sites are possible regulatory sites for the interaction of ptpip51 with 14 - 3 - 3beta , thus being able to influence the mapk pathway by interaction with raf-1 .
phosphorylation of serine 46 in ptpip51 results in augmented interaction with raf-1 via 14 - 3 - 3 and thus in increased erk1/2 phosphorylation , promoting the mapk pathway [ 12 , 17 ] .
pka is known to induce lipolysis through the mapk pathway [ 18 , 19 ] .  
the insulin receptor induces tyrosine phosphorylation of ptpip51 at tyrosine 176 , which leads to reduced interaction of ptpip51 with 14 - 3 - 3beta and raf-1 , thereby decreasing the mapk pathway activity .
analysis of the ptpip51 interaction profile of adipocytes displayed high interaction levels with the insulin receptor in hfd animals .
interaction levels were reduced by 30% in sd and hfdt and by 50% in sdt animals , compared to hfd animals .  
high - fat feeding increases the lipogenic activity in adipose tissue [ 21 , 22 ] .
the reduced ptpip51-ir interaction in trained groups compared to the corresponding untrained groups suggests that ptpip51 is especially involved in the processes of insulin induced lipogenesis or in its antilipolytic action .
interaction levels between ptpip51 and pka did not show significant differences in sd , hfd , and hfdt animals . in sdt animals ,
but , as pka also interacts with the hormone - sensitive lipase ( hsl ) , a change of the interaction partner of pka away from ptpip51 under these preconditions is assumable .
the activities of both pka and hsl are increased in endurance - trained animals [ 24 , 25 ] .  
our experiments suggest that ptpip51 could mediate between lipogenesis and lipolysis by switching between the lipogenic insulin pathway and the lipolytic pka pathway .
further experiments need to be conducted in order to prove our hypothesis and to determine the magnitude in which ptpip51 engages in adipocyte metabolism .
 
 aims . 
 our previous experiments revealed an association of ptpip51 ( protein tyrosine phosphatase interacting protein 51 ) with the insulin signalling pathway through ptp1b and 14 - 3 - 3beta . 
 we aimed to clarify the role of ptpip51 in adipocyte metabolism . 
 methods . 
 four groups of ten c57bl/6 mice each were used . 
 two groups were fed a standard diet ; two groups were fed a high - fat diet . 
 two groups ( one high - fat diet and one standard diet ) were submitted to endurance training , while the remaining two groups served as untrained control groups . 
 after ten weeks , we measured glucose tolerance of the mice . 
 adipose tissue samples were analyzed by immunofluorescence and duolink proximity ligation assay to quantify interactions of ptpip51 with either insulin receptor ( ir ) or pka . 
 results . ptpip51 and the ir and ptpip51 and pka , respectively , were colocalized in all groups . 
 standard diet animals that were submitted to endurance training showed low ptpip51-ir and ptpip51-pka interactions . 
 the interaction levels of both the ir and pka differed between the feeding and training groups . 
 conclusion . 
 ptpip51 might serve as a linking protein in adipocyte metabolism by connecting the ir - triggered lipogenesis with the pka - dependent lipolysis . ptpip51 
 interacts with both proteins , therefore being a potential gateway for the cooperation of both pathways . 
1. Introduction
2. Material and Methods
3. Results
4. Discussion