Cardiac contractile dysfunction in insulin-resistant rats fed a high-fat diet is associated with elevated CD36-mediated fatty acid uptake and esterification Aims/hypothesis Changes in cardiac substrate utilisation leading to altered energy metabolism may underlie the development of diabetic cardiomyopathy . We studied cardiomyocyte substrate uptake and utilisation and the role of the fatty acid translocase CD36 in relation to in vivo cardiac function in rats fed a high-fat diet -LRB- HFD -RRB- . Introduction Diabetic cardiomyopathy -LRB- DCM -RRB- is a common complication in type 2 diabetes -LSB- 1 -RSB- . Changes in myocardial energy metabolism , due to altered substrate handling , characterise diabetes-related heart disease and DCM -LSB- 1 -RSB- . Studies in rodent models , including rats fed a high-fat diet -LRB- HFD -RRB- -LSB- 2 -RSB- , leptin-deficient and - resistant animals -LSB- 3 -- 6 -RSB- and transgenic mice with cardiac-restricted peroxisome proliferator activated receptor α -LRB- PPARα -RRB- overexpression -LSB- 7 -RSB- , indicate that intramyocardial accumulation of triacylglycerol metabolites -LRB- lipotoxicity -RRB- and myocardial insulin resistance may underlie diabetes-related cardiac dysfunction -LSB- 1 , 8 -RSB- . Among the putative causes of myocardial triacylglycerol accumulation are elevations in long-chain fatty acid -LRB- LCFA -RRB- uptake -LSB- 6 , 7 , 9 -RSB- . In the heart , approximately 50 % of LCFA uptake is mediated by the fatty acid translocase CD36 -LSB- 10 , 11 -RSB- . Under physiological conditions , both insulin and contraction stimulate LCFA uptake by inducing the translocation of CD36 from an intracellular pool to the sarcolemma , illustrating that CD36 is also directly involved in the dynamic utilisation of LCFA by cardiomyocytes . This acute translocation of CD36 involves activation of phosphatidylinositol 3 ′ - kinase -LRB- PI3K -RRB- / protein kinase B -LRB- PKB/Akt -RRB- - and AMP-kinase -LRB- AMPK -RRB- - dependent signalling cascades in response to insulin and contraction , respectively -LSB- 11 -RSB- . Interestingly , CD36 deletion rescues the cardiac dysfunction , metabolic abnormalities and myocardial triacylglycerol accumulation observed in transgenic mice with cardiac-restricted PPARα overexpression -LSB- 7 -RSB- , suggesting a critical role for CD36 in the development of diabetes-related heart disease -LSB- 12 -RSB- . We hypothesised that long-term alterations in the functional pool of CD36 lead to myocardial accumulation of -LRB- toxic -RRB- lipid metabolites and insulin resistance and contribute to the development of diabetes-related heart disease . To examine this , we first measured in vivo cardiac dimensions by echocardiography in rats exposed to a high-fat diet -LRB- HFD -RRB- , which impairs myocardial insulin signalling -LSB- 2 , 13 -RSB- . Subsequently , isolated cardiomyocytes were used to determine changes in substrate uptake and LCFA metabolism under basal conditions and after incubations with insulin and with oligomycin ; the latter compound induces a contraction-like elevation of intracellular AMP levels and concomitant activation of AMPK -LSB- 14 -RSB- . Finally , we assessed whether the observed alterations could be ascribed to CD36 in hearts from rats on the HFD by analysing the effects of the CD36 inhibitor sulfo-N-succinimidyloleate -LRB- SSO -RRB- on myocardial LCFA uptake and utilisation in isolated cardiomyocytes , and by studying the expression and subcellular localisation of CD36 in cardiac ventricular tissue . Materials and methods Animals The investigation conformed to the Guide for the Care and Use of Laboratory Animals published by the NIH -LRB- NIH Publication No. 85-23 , revised 1996 -RRB- and was approved by the VU University Medical Centre Animal Care Committee . Adult male Wistar WU rats -LRB- n = 31 ; mean body weight 361 ± 24 g -RRB- were purchased from Harlan CBP -LRB- Horst , the Netherlands -RRB- , and fed an HFD or an isocaloric low-fat diet -LRB- LFD -RRB- -LSB- 2 -RSB- . At week 8 , rats fasted overnight underwent an OGTT , and post-load blood glucose levels were determined 15 , 30 , 60 , 90 and 120 min after ingestion of 2 g glucose/kg body weight -LSB- 2 -RSB- . After 10 weeks on the diet , blood was collected for determination of glucose and insulin levels . Rats were then anaesthetised with an intraperitoneal injection of sodium pentobarbital , and hearts were removed for the isolation of cardiomyocytes -LSB- 15 -RSB- . Diets Experimental diets were obtained from Hope Farms -LRB- Woerden , the Netherlands ; HFD , catalogue no. 4148.02 ; LFD , catalogue no. 4148.01 -RRB- . The LFD consisted of 8 % by weight of total fat , 22 % by weight of protein and 60 % by weight of carbohydrate ; the HFD contained 25 % by weight of fat , 32 % by weight of protein and 25 % by weight of carbohydrate , as well as more palmitate -LRB- 91.12 g/kg -RRB- and oleate -LRB- 100.24 g/kg -RRB- compared with the LFD -LRB- 29.12 and 32.08 g/kg , respectively -RRB- . In the group fed the HFD , 50.4 % of the ingested calories were derived from fat compared with 16.4 % in the group fed the LFD . In vivo cardiac function Non-invasive transthoracic echocardiograms were recorded at heart rates of 350 -- 400 beats per minute in rats anaesthetised with 1.5 % isoflurane in a mixture of N2O/O2 -LRB- 1/2 , vol/vol -RRB- -LSB- 16 -RSB- before and 8 weeks after initiation of the diet by an Aloka echo machine -LRB- ProSound SSD-4000 -RRB- using a 13-MHz linear interfaced array transducer . M - and B-mode images were obtained in the parasternal long - and short-axis views of the left ventricle -LRB- LV -RRB- at mid-papillary level . At end systole -LRB- ES -RRB- and end diastole -LRB- ED -RRB- , LV lumen diameter -LRB- D -RRB- , LV ventricular diameter -LRB- VD -RRB- and posterior -LRB- PWT -RRB- and interventricular septum -LRB- IVSWT -RRB- wall thicknesses were determined for three cardiac cycles and averaged . The LV dimensions were used to calculate systolic parameters -LSB- 17 -RSB- : fractional shortening , where EDD is end diastolic diameter and ESD is end systolic diameter ; end diastolic volume -LRB- EDV -RRB- = -LSB- EDD -RSB- 3 ; end systolic volume -LRB- ESV -RRB- = -LSB- ESD -RSB- 3 ; and ejection fraction . LV mass -LRB- LVM -RRB- was determined according to the uncorrected cube assumption : , where 1.05 is the specific gravity of the myocardium -LSB- 18 -RSB- . Plasma and tissue determinations Blood glucose was measured from tail bleeds using a glucose analyser -LRB- HemoCue , Angelholm , Sweden -RRB- . Plasma insulin was measured using ELISA -LRB- EZRMI-13K ; Linco Research , St Charles , MO , USA -RRB- . Myocardial triacylglycerol content was determined in ventricular lysates from a separate set of animals as described in -LSB- 2 -RSB- . Treatment of isolated cardiomyocytes Cardiomyocytes were prepared using a Langendorff perfusion system as described in -LSB- 15 , 19 -RSB- . Isolated cardiomyocytes were incubated for 30 min at room temperature with 0.5 % DMSO or SSO -LSB- 9 -RSB- . The CD36-specific inhibitor SSO -LSB- 20 -RSB- was used to investigate the involvement of CD36 in basal LCFA uptake . SSO was applied at a concentration of 0.4 mmol/l , which effectively inhibits CD36 -LSB- 15 , 21 -RSB- . Then , cells were washed and incubation was continued for 15 min at 37 °C with continuous shaking . Thereafter , the DMSO-treated cardiomyocytes were kept for another 15 min at 37 °C and either kept untreated -LRB- basal -RRB- or incubated with insulin or oligomycin . To achieve optimal stimulation of glucose and LCFA uptake -LSB- 22 -RSB- , insulin and oligomycin were used at a concentration of 10 nmol/l and 30 μmol/l , respectively . At this concentration , the ATP-synthase inhibitor oligomycin induces an increase in the intracellular AMP/ATP ratio , thereby activating AMPK-mediated signalling pathways without inhibiting oxygen consumption -LSB- 14 -RSB- . Glucose uptake , palmitate uptake and oxidation , and incorporation into intracellular lipid pools Following agonist treatment , -LSB- 3H -RSB- -2-deoxyglucose and -LSB- 1-14C -RSB- palmitate uptake were simultaneously determined during a 3-min incubation -LSB- 15 -RSB- . Rates of -LSB- 1-14C -RSB- palmitate oxidation -LRB- measured as the production of 14CO2 -RRB- and esterification -LRB- measured as incorporation of the 14C label into intracellular phospholipids -LSB- PL -RSB- , and triacylglycerol -RRB- were determined after a 20-min incubation -LSB- 9 -RSB- . Western blotting Phosphorylation of proline-rich Akt-substrate 40 -LRB- PRAS40-Thr246 -RRB- and acetyl-coenzyme A carboxylase-Ser79 -LRB- ACC-Ser79 -RRB- was determined in homogenates of cardiomyocytes by western blotting using phospho-specific antibodies -LSB- 9 , 13 -RSB- . PKB/Akt-Ser 473 phosphorylation was determined in ventricular homogenates as described in -LSB- 2 -RSB- . Expression levels of CD36 were determined by western blotting using the monoclonal CD36 MO25 antibody -LSB- 23 -RSB- . Immunoblots were quantified by densitometric analysis of the films -LSB- 2 -RSB- . Cardiac morphology and subcellular localisation of CD36 Cardiac ventricular tissue collected in a previous study -LSB- 2 -RSB- was routinely embedded in paraffin -LRB- Histowax ; Leica Microsystems , Wetzlar , Germany -RRB- . Sections -LRB- 5 μm -RRB- were cut , and mounted on slides coated with 3-aminopropyltriethoxisilane -LRB- Menzal , Darmstadt , Germany -RRB- . After deparaffinisation and rehydration , the slides were used for determination of cardiomyocyte cross-sectional area or immunohistochemical staining of CD36.Cross-sectional area was determined in randomly chosen fields in haematoxylin/eosin-stained slides using NIH image analysis software -LRB- National Institutes of Health , Bethesda , MD , USA -RRB- for 20 -- 30 cells per heart and normalised to sarcomere length -LSB- 24 -RSB- . For CD36 localisation , sections were rinsed extensively with Tris-buffered saline -LRB- TBS ; 50 mmol/l Tris -- Cl , 500 mmol/l NaCl , pH 7.6 -RRB- , and incubated overnight with the monoclonal CD36 MO25 antibody -LSB- 23 -RSB- diluted 1:2,000 in TBS containing 0.5 % Triton X-100 , pH 7.6 at 4 °C . After several washes in TBS , sections were incubated for 1.5 h at room temperature with biotinylated anti-mouse IgG -LRB- Vector Laboratories , Burlingame , CA , USA -RRB- , diluted 1:250 in TBS , followed by incubation for 1 h at room temperature with avidin -- biotin -- peroxidase -LRB- 1:400 ; ABC Elite kit ; Vector Laboratories -RRB- . Tissue-bound peroxidase was visualised with the 3,3 ′ - diaminobenzidine tetrahydrochloride -LRB- DAB -RRB- chromogen reaction -LRB- 7.5 mg DAB , 5 μl 30 % H2O2 in 15 ml 50 mmol/l Tris -- Cl , pH 7.6 -RRB- for 10 min at room temperature . Sections were then rinsed with distilled water , dehydrated in a graded series of ethanol , cleared in xylene , and coverslipped with Entellan -LRB- Merck , Darmstadt , Germany -RRB- . Sections were analysed using a Leica DM-LB light microscope -LRB- Leica , Rijswijk , the Netherlands -RRB- . Digital images were taken at × 200 magnification using a Leica DC500 digital camera , and quantified using Quantity One software -LRB- Bio-Rad Laboratories , Veenendaal , the Netherlands -RRB- . Statistical analysis Unless indicated otherwise , data are expressed as means ± SE for the indicated number of animals -LRB- n -RRB- . Statistical analysis was performed in SPSS for Mac OS X version 11.0.4 -LRB- SPSS Inc. , Chicago , IL , USA -RRB- . Differences between the LFD and HFD groups were determined using independent t tests . The paired sample t test was used to define the effect of agonist incubations on cardiomyocyte preparations . p < 0.05 was considered as statistically significant . Results HFD induces cardiac contractile dysfunction Feeding rats isocaloric LFD or HFD diets for 8 weeks induced similar weight gains in the two groups -LRB- Table 1 -RRB- , but mild glucose intolerance , as determined by an oGTT , in HFD - vs LFD-rats -LRB- p < 0.05 ; Fig. 1a -RRB- . Figure 1b shows the LV dimensions at the start and 8 weeks after initiation of the diet interventions . Baseline parameters were similar in the two groups -LRB- Table 1 -RRB- . After 8 weeks on the diet , LVM , ED-VD , EDD and ED-IVSWT showed comparable increases in LFD and HFD hearts -LRB- all p < 0.05 -RRB- , whereas ED-PWT was unaffected by the diet -LRB- Table 1 -RRB- . In the ES phase , both ES-PWT and ES-IVSWT were increased in LFD hearts only -LRB- both p < 0.005 ; Table 1 -RRB- . ES-VD was similarly affected by LFD and HFD feeding . ESD was increased in LV from HFD-fed vs LFD-fed rats -LRB- p < 0.01 ; Table 1 -RRB- . FS % was increased in LFD hearts -LRB- p < 0.05 -RRB- , and decreased in HFD hearts during the course of the diet intervention -LRB- p < 0.02 ; Table 1 -RRB- . Accordingly , EF % was decreased in HFD compared with LFD hearts -LRB- p < 0.05 ; Table 1 -RRB- . Collectively , these data show that HFD induced cardiac contractile dysfunction in rats . Table 1In vivo cardiac characteristics of rats before and after 8 weeks on an HFD or a LFD StartLFDHFD -LRB- n = 24 -RRB- -LRB- n = 8 -RRB- -LRB- n = 16 -RRB- Physiological parameters Body weight -LRB- g -RRB- 295 ± 4476 ± 8 * 463 ± 8 * Left ventricular mass -LRB- mg -RRB- 637 ± 15890 ± 18 * 882 ± 32 * LV diastolic parameters Posterior wall thickness -LRB- mm -RRB- 1.63 ± 0.031.80 ± 0.071.71 ± 0.05 Lumen diameter -LRB- mm -RRB- 6.92 ± 0.077.55 ± 0.20 * 7.68 ± 0.10 * Interventricular septum wall thickness -LRB- mm -RRB- 1.37 ± 0.031.67 ± 0.08 * 1.54 ± 0.04 * Ventricular diameter -LRB- mm -RRB- 9.80 ± 0.0610.86 ± 0.13 * 10.89 ± 0.12 * LV systolic parameters Posterior wall thickness -LRB- mm -RRB- 2.93 ± 0.063.45 ± 0.12 * 3.00 ± 0.08 ** Lumen diameter -LRB- mm -RRB- 3.41 ± 0.103.32 ± 0.254.07 ± 0.12 * , ** Interventricular septum wall thickness -LRB- mm -RRB- 2.51 ± 0.052.85 ± 0.09 * 2.67 ± 0.06 Ventricular diameter -LRB- mm -RRB- 8.81 ± 0.079.82 ± 0.12 * 9.81 ± 0.10 * Fractional shortening -LRB- % -RRB- 50.8 ± 1.356.3 ± 2.4 * 47.1 ± 1.7 * , ** Ejection fraction -LRB- % -RRB- 87.5 ± 1.091.1 ± 1.484.9 ± 1.0 * , ** Data are means ± SE * p < 0.05 vs start ** p < 0.01 vs LFDFig . 1a Blood glucose levels after an oral glucose load in rats fed the HFD -LRB- closed circles ; n = 16 -RRB- and the LFD -LRB- open circles ; n = 8 -RRB- . Data are means ± SE . b Representative echocardiographic M-mode images from at least three cardiac contractile cycles at the start of the diet intervention and 8 weeks after initiation of the LFD and HFD , respectively Insulin action , but not AMPK signalling , is impaired in HFD cardiomyocytes We used cardiomyocytes to unravel the diet-induced molecular alterations in energy substrate uptake and intracellular signalling pathways . Cardiomyocytes were isolated from a separate group of rats , fed either the LFD or the HFD under identical experimental conditions . At killing , body , heart and liver weights were similar between LFD - and HFD-fed rats , whereas perirenal fat pad weight was 50 % higher in HFD - than in LFD-fed rats -LRB- p < 0.001 ; Table 2 -RRB- . Furthermore , HFD feeding increased blood glucose levels -LRB- p < 0.02 -RRB- and lowered plasma insulin levels -LRB- p < 0.05 -RRB- . Table 2Characteristics of rats after 10 weeks on a high - or low-fat diet LFDHFD -LRB- n = 14 -RRB- -LRB- n = 17 -RRB- Body composition Body weight at killing -LRB- g -RRB- 465 ± 5472 ± 10 Heart weight -LRB- % body weight -RRB- 0.445 ± 0.0070.431 ± 0.012 Liver weight -LRB- % body weight -RRB- 3.10 ± 0.073.02 ± 0.08 Perirenal fat pad weight -LRB- % body weight -RRB- 1.63 ± 0.082.43 ± 0.14 * Plasma characteristics Blood glucose -LRB- mmol/l -RRB- 5.50 ± 0.085.78 ± 0.08 ** Plasma insulin -LRB- pmol/l -RRB- 269 ± 18216 ± 16 *** Data are means ± SE * p < 0.001 vs LFD ** p < 0.02 vs LFD *** p < 0.05 vs LFDBasal 2-deoxyglucose glucose uptake rates were 7.3 ± 0.8 and 11.0 ± 1.6 nmol min − 1 -LSB- g wet cell mass -RSB- -1 C in LFD and HFD cells , respectively -LRB- p = 0.063 ; Fig. 2 -RRB- . Insulin stimulated 2-deoxyglucose uptake 3.7-fold in LFD cardiomyocytes and 2.2-fold in HFD cardiomyocytes -LRB- both p < 0.001 vs basal , p < 0.02 HFD vs LFD -RRB- , indicating reduced myocardial insulin responsiveness . Oligomycin had similar stimulatory effects on 2-deoxyglucose uptake in HFD and LFD cardiomyocytes -LRB- HFD , 1.6-fold stimulation ; LFD , 1.7-fold stimulation ; both p < 0.05 vs basal -RRB- . Fig. 2Rates of 2-deoxyglucose uptake in cardiomyocytes from rats fed the LFD -LRB- open bars -RRB- and the HFD -LRB- filled bars -RRB- . Cardiomyocytes were incubated with DMSO -LRB- Basal -RRB- , insulin -LRB- INS -RRB- or oligomycin -LRB- Oli -RRB- before measurement of 2-deoxyglucose uptake . Data are expressed as wet cell mass and are means ± SE ; n = 8 . * p < 0.02 , agonist effect ; # p < 0.02 , diet effectActivation of phosphatidylinositol 3 ′ - kinase -LRB- PI3K -RRB- / protein kinase B -LRB- PKB/Akt -RRB- - and AMP-kinase -LRB- AMPK -RRB- - dependent signalling cascades has been implicated in the stimulation of glucose uptake in response to insulin and oligomycin , respectively . We studied whether changes in the activation of these signalling pathways paralleled the observed alterations in glucose uptake rates in HFD cells . Therefore , phosphorylation of PRAS40-Thr246 and ACC-Ser79 were examined as respective distal determinants for PKB/Akt and AMPK activity -LSB- 13 , 25 -RSB- , as the presence of bovine serum albumin -LRB- mol . mass 66 kDa -RRB- in the cardiomyocyte homogenates interfered with direct measurement of PKB/Akt -LRB- mol . mass 64 kDa -RRB- and AMPKα phosphorylation -LRB- mol . mass 62 kDa -RRB- . Basal PRAS40-Thr246 phosphorylation was 1.7-fold higher in HFD than in LFD cells -LRB- p < 0.05 ; Fig. 3a , b -RRB- . Insulin stimulated PRAS40-Thr246 phosphorylation 12.0-fold in LFD cells -LRB- p < 0.001 -RRB- and 4.0-fold in HFD cells -LRB- p < 0.001 -RRB- , thus confirming reduced insulin responsiveness . Oligomycin did not affect PRAS40-Thr246 phosphorylation . Fig. 3Immunoblots -LRB- a , c -RRB- and quantification of PRAS40-Thr246 -LRB- b -RRB- and ACC-Ser79 -LRB- d -RRB- phosphorylation after incubation of cardiomyocytes from LFD - and HFD-fed rats with DMSO -LRB- Basal -RRB- , insulin -LRB- INS -RRB- or oligomycin -LRB- Oli -RRB- . Data are means ± SE ; n = 5 . * p < 0.05 , agonist effect ; # p < 0.05 , diet effectACC-Ser79 phosphorylation was measured as a determinant of AMPK activity -LSB- 25 -RSB- . Basal levels of Ser79-phosphorylated ACC were similar in LFD and HFD cells -LRB- Fig. 3c , d -RRB- . Insulin had no effect on ACC-Ser79 phosphorylation , whereas oligomycin stimulated ACC-Ser79 phosphorylation in LFD and HFD cells to a similar extent -LRB- HFD , 3.0-fold stimulation ; LFD , 2.9-fold stimulation ; both p < 0.001 vs basal -RRB- , indicating that AMPK signalling was not affected by high-fat feeding . HFD feeding results in increased CD36-mediated LCFA uptake Basal rates of LCFA uptake were increased 1.4-fold -LRB- p < 0.001 -RRB- in HFD compared with LFD cells -LRB- Fig. 4 -RRB- . The CD36 inhibitor SSO reduced basal LCFA uptake by 44 % -LRB- p < 0.001 -RRB- and 23 % -LRB- p < 0.05 -RRB- in HFD and LFD cardiomyocytes , respectively . Notably , the absolute rates of LCFA uptake were similar between SSO-treated LFD and HFD cells -LRB- Fig. 4 -RRB- , suggesting that the increased basal LCFA uptake rates in HFD cells can be ascribed to changes in the sarcolemmal pool of CD36 . Insulin stimulated LCFA uptake 1.3-fold in LFD cells -LRB- p < 0.02 vs basal -RRB- but had no effect in HFD cells -LRB- p < 0.02 vs LFD ; Fig. 4 -RRB- . Oligomycin increased LCFA uptake in LFD and HFD cardiomyocytes to the same extent -LRB- Fig. 4 ; both p < 0.05 vs basal -RRB- . Fig. 4Rates of palmitate uptake -LRB- a -RRB- , oxidation -LRB- b -RRB- and esterification into intracellular triacylglycerols and phospholipids -LRB- c -RRB- in cardiomyocytes from LFD-fed rats -LRB- open bars -RRB- and HFD-fed rats -LRB- filled bars -RRB- . Cardiomyocytes were incubated with DMSO -LRB- Basal -RRB- , insulin -LRB- INS -RRB- , oligomycin -LRB- Oli -RRB- or SSO before measurement of palmitate uptake . Data are wet cell mass and are means ± SE ; n = 8 . * p < 0.05 , agonist effect ; # p < 0.05 diet effectWe next examined the metabolic fate of LCFA following their uptake by HFD and LFD cardiomyocytes . Basal rates of LCFA oxidation were reduced by 22 % in HFD relative to LFD cells -LRB- Fig. 4b ; p < 0.01 -RRB- . SSO further lowered cardiomyocyte LCFA oxidation rates by 66 % -LRB- p < 0.005 -RRB- and 31 % -LRB- p < 0.05 -RRB- in LFD and HFD cells , respectively , but absolute rates of LCFA oxidation were comparable in SSO-treated LFD and HFD cells.The incorporation rate of LCFA into triacylglycerol was increased 1.4-fold -LRB- p < 0.05 -RRB- in HFD compared with LFD cells under basal conditions -LRB- Fig. 4c -RRB- . SSO reduced triacylglycerol formation in both LFD and HFD cells -LRB- both p < 0.05 vs basal -RRB- . Notably , the absolute triacylglycerol esterification rates in SSO-treated cells did not differ between HFD - and LFD-fed rats . Also , the incorporation of LCFA into phospholipids under basal conditions was increased 1.5-fold in HFD vs LFD cells -LRB- p < 0.05 ; Fig. 4c -RRB- , and was reduced by SSO in both LFD and HFD cells -LRB- both p < 0.05 vs basal -RRB- . Myocardial triacylglycerol content was 181 ± 50 μg/mg protein in LFD rats and 330 ± 114 μg/mg protein in HFD rats -LRB- p < 0.02 -RRB- -LSB- 2 -RSB- . HFD induces cardiomyocyte hypertrophy and alters the subcellular localisation of CD36 The inhibitory effect of SSO suggests that the increased basal LCFA uptake and esterification rates in HFD cells can be ascribed to changes in the sarcolemmal pool of CD36 . Western blot analysis indicated that the levels of CD36 were similar between HFD and LFD hearts -LRB- Fig. 5a -RRB- . Immunohistochemical staining of cardiac LV tissue indicated cardiomyocyte hypertrophy , as reflected by an increased cross-sectional area -LRB- p < 0.05 vs LFD ; Table 3 -RRB- , and showed a more abundant presence of CD36 at the sarcolemmal membrane , particularly at the intercalated discs , in HFD hearts compared with LFD hearts -LRB- p < 0.002 vs LFD ; Fig. 5b , c and Table 3 -RRB- . Fig. 5Expression and subcellular localisation of CD36 . Expression of CD36 in ventricular lysates of HFD and LFD rats . Ponceau S staining confirmed that the filters contained equal amounts of ventricular extracts -LRB- a -RRB- . Immunohistochemical staining for CD36 localisation of cardiac LV tissue sections from rats fed an LFD -LRB- b , d -RRB- or HFD -LRB- c , e -RRB- for 8 weeks . Rats received an i.p. injection of saline -LRB- b , c -RRB- or insulin -LRB- d , e -RRB- 30 min before killing . Photographs are representative of two independent experiments performed on three rats per experimental group . Arrows indicate intercalated discs . Scale bar indicates 25 μmTable 3Cardiac morphology and subcellular localisation of CD36 LFDHFD -LRB- n = 6 -RRB- -LRB- n = 8 -RRB- Morphology Cardiomyocyte cross-sectional area normalised to sarcomere length -LRB- μm2 -RRB- 214 ± 3.5276 ± 23 * CD36 localisation Sarcolemma Saline -LRB- % -RRB- 61.7 ± 374.8 ± 1 ** Insulin -LRB- % -RRB- 75.9 ± 4 *** 78.2 ± 3 Cytoplasm Saline -LRB- % -RRB- 38.3 ± 325.2 ± 1 ** Insulin -LRB- % -RRB- 24.1 ± 4 *** 21.8 ± 3Data are means ± SE * p < 0.05 vs LFD ** p < 0.002 vs LFD *** p < 0.01 vs salineAs insulin stimulated LCFA uptake in isolated cardiomyocytes -LRB- Fig. 4 -RRB- , we also examined the subcellular localisation of CD36 in hearts isolated 30 min following an intraperitoneal injection with insulin -LRB- 10 U/kg body weight -RRB- -LSB- 2 -RSB- . Figure 5d shows that in vivo insulin treatment increased CD36 immunoreactivity at the intercalated discs in LFD hearts -LRB- p < 0.01 vs saline ; Table 3 -RRB- . In HFD hearts , insulin did not further recruit CD36 to the sarcolemma -LRB- Fig. 5e -RRB- . HFD-induced CD36 redistribution to the sarcolemma precedes the onset of cardiac contractile dysfunction and associates with elevated basal phosphorylation of PKB/Akt We next analysed whether HFD-induced alterations in CD36 localisation precede the onset of cardiac contractile dysfunction , using rats fed an LFD or HFD for 4 weeks . This period was chosen based on serial echocardiographic measurements , which revealed a tendency towards decreased cardiac contractile function 4 weeks after initiation of the diet intervention . Specifically , FS % was 4.2 % lower -LRB- p = 0.07 -RRB- and EF % was 3.3 % lower -LRB- p = 0.10 -RRB- in HFD vs LFD hearts after 4 weeks on the diet -LRB- data not shown -RRB- , compared with the 9.2 and 6.2 % decreases observed after 8 weeks of the feeding regime -LRB- both p < 0.05 ; Table 1 -RRB- . Despite the absence of significant effects on cardiac function after 4 weeks of HFD feeding , immunohistochemical staining of cardiac LV tissue showed that the abundance of CD36 at the sarcolemmal membrane was already markedly increased -LRB- Fig. 6a , b -RRB- to levels almost similar to those observed after 8 weeks of HFD feeding -LRB- Fig. 5b , c -RRB- . Fig. 6Effect of the duration of HFD feeding on CD36 localisation and phosphorylation of PKB/Akt . Immunohistochemical staining for CD36 localisation of cardiac LV tissue sections from saline-injected rats fed an LFD -LRB- a -RRB- or HFD -LRB- b -RRB- for 4 weeks . Photographs are representative of two or three independent experiments performed on six rats per experimental group . The arrows indicate intercalated discs . The scale bar indicates 25 μm . c Immunoblot and quantification d of PKB/Akt-Ser 473 phosphorylation levels in ventricular homogenates from saline-injected rats fed an LFD -LRB- open bars -RRB- or HFD -LRB- filled bars -RRB- for 4 and 8 weeks , respectively . Data are means ± SE , n = 8 . * p < 0.05 , diet effectFinally , we analysed whether HFD feeding induced changes in the activity of signalling pathways implicated in CD36 trafficking . In the healthy myocardium , CD36 is stored in at least two endosomal storage pools that are regulated by AMPK and PKB/Akt , respectively -LSB- 26 -RSB- . As insulin does not further recruit CD36 to the sarcolemma in HFD hearts -LRB- Fig. 5e -RRB- , we proposed that increases in basal activity of PKB/Akt may underlie the redistribution of CD36 in HFD hearts . Phosphorylation of PKB/Akt was determined in LV homogenates of the same group of saline-injected rats in which CD36 localisation was measured . As shown in Fig. 6c , d , basal phosphorylation of PKB/Akt was 1.7 - and 2.6-fold higher after 4 and 8 weeks of HFD feeding , respectively -LRB- both p < 0.05 -RRB- . Collectively , these results indicate that increased activity of PKB/Akt may underlie CD36 redistribution to the sarcolemma , and that these events precede the LV dysfunction induced by HFD feeding . Discussion Here we report that HFD feeding induces cardiac contractile dysfunction in rats and that this is associated with a permanent relocation of CD36 to the sarcolemma . The continuous presence of CD36 at the sarcolemmal membrane results in enhanced rates of LCFA uptake and subsequent esterification . We propose that this contributes to a decrease in myocardial insulin action and the development of diabetes-related heart disease . In addition , we show that AMPK-mediated responses are not affected by the composition of the diet . A key observation in this study is that the alterations in cardiac contractile function in HFD hearts was associated with a continuous presence of CD36 at the sarcolemmal membrane . The present study provides the first morphological evidence for translocation of CD36 to the sarcolemmal membrane . Importantly , the amount of sarcolemmal CD36 closely correlated with enhanced LCFA uptake rates in isolated cardiomyocytes . The observation that SSO inhibited LCFA uptake in HFD cardiomyocytes to the same residual levels as measured in LFD cardiomyocytes provides further pharmacological evidence that the enhanced flux of LCFA in the heart of HFD-fed rats is a direct consequence of the relocalisation of CD36 to the sarcolemmal membrane . Previously , a redistribution of CD36 to both subsarcolemmal and intramyofibrillar mitochondria has also been observed -LSB- 27 , 28 -RSB- . Can the observed CD36 immunoreactivity be ascribed to subsarcolemmal mitochondria rather that the sarcolemma itself ? Stimuli inducing translocation of CD36 to the mitochondria are expected not to discriminate between the subsarcolemmal and the intramyofibrillar mitochondria . Hence , if a stimulus or condition , in this case insulin or HFD feeding , were to increase the subsarcolemmal CD36 content , one would expect a similar increase in intramyofibrillar CD36 content . This , however , could not be confirmed in the immunohistochemical experiments . Furthermore , biochemical fractionations performed in previous studies substantiate the idea that a significant fraction of CD36 is present at the sarcolemma of hearts from insulin-resistant rats , and that only a minor portion of CD36 is found within the mitochondrial fractions -LSB- 9 , 22 -RSB- . While insulin has been shown to translocate CD36 to the sarcolemma -LSB- 9 , 22 -RSB- , insulin-mediated CD36 translocation to the mitochondria has never been reported and even seems counterintuitive . Collectively , the available evidence strongly supports the idea that the observed CD36 immunoreactivity can be ascribed to CD36 located at the sarcolemma rather than in subsarcolemmal mitochondria . The combined data of this and our earlier study on obese Zucker rats -LSB- 9 -RSB- suggest that relocalisation of CD36 is a general phenomenon in insulin-resistant hearts , and raises the question of what mechanism underlies the continuous presence of CD36 at the sarcolemmal membrane in HFD hearts . In the healthy myocardium , CD36 is stored in at least two endosomal storage pools that are regulated by AMPK and PI3K/PKB/Akt , respectively -LSB- 26 -RSB- . Activation of AMPK is critical for contraction - and oligomycin-mediated CD36 translocation , whereas PI3K/PKB/Akt is critical for insulin-mediated CD36 trafficking -LSB- 10 , 14 , 26 -RSB- . Basal phosphorylation of PKB/Akt and its distal target , PRAS40 , were elevated in HFD compared with LFD hearts , whereas AMPK activity was not affected by the diet . Furthermore , in cells from HFD-fed rats , the stimulatory effects of insulin on LCFA uptake , CD36 translocation and phosphorylation of PKB/Akt and PRAS40 were abrogated , whereas oligomycin-induced AMPK activation and LCFA uptake was unimpaired between cardiomyocytes from LFD - and HFD-fed rats . Although we can not exclude a contribution of other kinases or HFD-induced alterations in the as yet undisclosed trafficking machinery regulating the internalisation of CD36 -LSB- 11 , 26 -RSB- , our observations do not argue against the suggestion that increases in PKB/Akt activity may contribute to the sustained sarcolemmal presence of CD36 in the heart of HFD-fed rats . Previously , we suggested that increased plasma insulin might contribute to CD36 relocalisation in hearts from obese Zucker rats -LSB- 9 -RSB- . As systemic hyperinsulinaemia was not observed in HFD-fed rats -LSB- 2 , 13 -RSB- , it remains interesting to examine whether changes in the activity of other -LRB- insulin-independent -RRB- regulators of PKB/Akt phosphorylation , such as PI3Kγ - and β2-adrenergic receptor signalling pathways , Ca2 + - calmodulin dependent kinase , protein phosphatase 2A and the sympathetic nervous system -LSB- 29 -RSB- , can be linked to the observed increase in PKB/Akt phosphorylation in HFD hearts . The metabolic and biochemical data were paralleled by in vivo cardiac functional changes . Importantly , HFD-induced cardiac contractile dysfunction does not seem to be linked to diet-related elevations in blood pressure . Circadian haemodynamic parameters were monitored using implanted telemetry devices in an experiment performed in a separate group of animals . We found a slight increase in night-time -LRB- activity-related -RRB- heart rate in HFD rats , but no changes in blood pressure even after 10 weeks of exposure to the diet -LRB- Electronic supplementary material Table 1 -RRB- . Others confirmed the absence of relevant hypertension after feeding rats a diet containing 74 % fat for 17 weeks -LSB- 30 -RSB- . This underscores the possibility that alterations in metabolism , rather than in haemodynamics , underlie the impairment in cardiac contractile function in HFD rats . The present study also suggests that HFD-induced alterations in the functional pool of CD36 may contribute to an imbalance in LCFA uptake and in oxidation and esterification rates in HFD vs LFD cells . Whereas LCFA uptake rates were increased 1.4-fold in HFD cells , the LCFA oxidation rates were not increased but rather modestly lower in HFD cells , while the rates of esterification into triacylglycerol and phospholipids were 1.4-fold higher in HFD vs LFD cardiomyocytes . Accordingly , triacylglycerol content was 1.9-fold increased in HFD compared with LFD hearts -LSB- 2 -RSB- . It seems unlikely that changes in malonylCoA levels underlie the reduced rates of LCFA oxidation in HFD hearts , as no diet-induced changes in ACC phosphorylation were found . Rather , degenerative changes in mitochondria , such as matrix dilution , cristolysis and mitochondria-associated lamellar bodies , have been observed in cardiomyocytes from HFD-fed rats -LSB- 2 -RSB- and have been linked to reduced oxidative capacity and lipid accumulation in skeletal muscle from patients with type 2 diabetes . However , further studies are required to examine whether these changes contribute to the reduced basal LCFA oxidation and increased LCFA esterification rates , or whether the extra LCFA taken up by HFD cells are stored as triacylglycerol and phospholipids as a consequence of HFD-induced changes in enzymes promoting LCFA esterification or inhibition of lipolysis . An important finding of this study is that the HFD-induced CD36 redistribution preceded the onset of cardiac contractile dysfunction . Although we can not unequivocally link CD36 redistribution to cardiac dysfunction , a recent report provided strong support for this notion as the absence of CD36 was found to prevent myocardial triacylglycerol accumulation in transgenic mice with cardiac PPARα overexpression , both under normal conditions and after HFD feeding -LSB- 12 -RSB- . Similarly , alterations in PKB/Akt activity have been linked to cardiac dysfunction -LSB- 31 -RSB- . Myocardial biopsy samples obtained from patients with advanced heart failure or dilated cardiomyopathy show increased basal phosphorylation of PKB/Akt -LSB- 32 , 33 -RSB- , and transgenic mouse models with constitutively activated PKB/Akt in the heart develop hypertrophy , decreased cardiac function and impaired recovery from ischaemia -- reperfusion injury -LSB- 31 , 34 -RSB- . Whereas chronic activation of PKB/Akt increases basal glucose uptake rates in the heart -LSB- 35 -RSB- , the effects on myocardial lipid metabolism have not been studied . Interestingly , PKB/Akt signalling has been linked to palmitate-induced beta cell lipotoxicity -LSB- 36 -RSB- . Based on this report and the findings described in this study , it would be of interest to analyse animals expressing chronically active PKB/Akt for translocation of CD36 to the sarcolemma and myocardial lipid accumulation . It seems plausible that an enhanced CD36-mediated LCFA uptake rate may contribute to the development of cardiac contractile dysfunction . An increased supply of LCFA may influence excitation -- contraction coupling as well as other processes linked to cellular Ca2 + handling -LSB- 37 , 38 -RSB- . Furthermore , triacylglycerol accumulation is strongly linked to cardiac insulin resistance and contractile dysfunction -LSB- 1 , 2 , 8 -RSB- . The dynamic equilibrium between triacylglycerol stores and triacylglycerol metabolites causes accumulation of ceramide and diacylglycerol during prolonged LCFA influx . Both ceramide and diacylglycerol have been implicated in the activation of serine/threonine kinases , such as PKC , Jun N terminal kinase -LRB- JNK -RRB- , and inhibitor of nuclear factor-κB kinase -LRB- IKK -RRB- , which counteract insulin signalling -LSB- 39 , 40 -RSB- . Notably , chronic myocardial activation of Akt in transgenic mice has also been linked to feedback inhibition of PI3K activity -LSB- 33 -RSB- and blunted insulin stimulation of glucose uptake -LSB- 35 -RSB- . Importantly , reduced insulin responsiveness reduces the ability of insulin to regulate substrate handling , e.g. metabolic inflexibility -LSB- 41 -RSB- . In this study and previous reports -LSB- 2 , 13 -RSB- , we show that HFD feeding reduces insulin responsiveness and impairs metabolic flexibility , as illustrated by the blunted effects of insulin on the stimulation of glucose uptake , phosphorylation of PRAS40 , LCFA uptake and translocation of CD36 to the sarcolemma . We conclude that HFD feeding in rats induces cardiac contractile dysfunction , which is preceded by relocation of CD36 to the sarcolemma , and elevated basal levels of phosphorylated PKB/Akt . The continuous presence of CD36 at the sarcolemma contributes to enhanced rates of fatty acid uptake , resulting in myocardial triacylglycerol accumulation and accompanying insulin resistance . Collectively , these data suggest that alterations in the subcellular localisation of CD36 may contribute to the development of diabetes-related heart disease and that CD36 may be a therapeutic target to prevent cardiac dysfunction and the development of heart failure in diabetes . Electronic supplementary material Below is the link to the electronic supplementary material . ESM Table 1 Heart rate and blood pressure of rats after 10 weeks on an HFD or a LFD -LRB- PDF 22 kb -RRB-

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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B I O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O B O O O O O O O O O O O O O B I O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

cd36
contractile dysfunction
diabetic cardiomyopathy
glucose uptake
insulin resistance


Fatty acid-induced mitochondrial uncoupling in adipocytes as a key protective factor against insulin resistance and beta cell dysfunction : do adipocytes consume sufficient amounts of oxygen to oxidise fatty acids ? To the Editor : The phenotype of patients with various forms of congenital lipodystrophy has indicated that proper storage of triacylglycerol in peripheral adipocytes is essential to prevent formation of ectopic fat deposits and development of type 2 diabetes -LSB- 1 -RSB- . A similar situation with induced peripheral lipodystrophy and elevated risk for type 2 diabetes is seen in patients undergoing highly active anti-retroviral therapy -LRB- HAART -RRB- . In these patients , there is a 30 -- 50 % decline in mitochondrial copy number as a result of HAART -LSB- 2 -RSB- . Based on these and other observations , we have recently put forward the hypothesis that one of the functions of mitochondria in adipose tissue is to prevent leakage of fatty acids into the circulation -LSB- 3 -RSB- . This mitochondrial activity attenuates the development of ectopic triacylglycerol deposits in the liver and other tissues that would otherwise contribute to the development of whole body insulin resistance and pancreatic beta cell damage . Mitochondria contribute to efficient confinement of fatty acids within adipocytes by oxidative removal of fatty acids , liberated from the triacylglycerol pool , by uncoupled beta oxidation . In addition , mitochondria may provide glyceroneogenic substrates , which contribute to re-esterification of fatty acids . As a net result , there are less fatty acids available to the circulation for redistribution to other compartments of the body -LSB- 3 -RSB- . A recent study provided supporting evidence for this concept by showing that the mitochondrial DNA copy number in human adipocytes is positively associated with lipogenesis in adipocytes -LSB- 4 -RSB- . Recently , Frayn et al. have argued against this biochemical mechanism -LSB- 5 -RSB- . They point out that fatty acid oxidation is not a major pathway in white adipocytes and that oxygen consumption by adipose tissue is insufficient to oxidise substantial amounts of fatty acids -LSB- 6 -RSB- . We fully agree with the point made by the authors that fatty acid oxidation by adipocytes is not a major pathway compared with muscle . However , we hold a different view on the quantitative interpretation of these data : in our model there is no need for a high rate of fatty acid oxidation in adipose tissue per time unit , as fatty acid redistribution from peripheral tissue to the liver is a slow process , taking several years . For instance , in individuals in whom mitochondrial copy number is acutely reduced as result of starting HAART , it takes 18 -- 24 months before redistribution of body fat becomes clinically manifest -LSB- 7 -RSB- . Therefore , the amount of fatty acids that undergoes redistribution per time unit is very small . Thus , mitochondria in adipocytes only need to remove a small amount of fatty acids per time unit to protect against redistribution of body fat . By using the oxygen consumption data of Frayn et al. and others -LSB- 5 , 6 -RSB- or using similar data from other studies -LSB- 8 , 9 -RSB- , one can calculate that oxygen consumption by adipose tissue is sufficient to prevent redistribution of > 1.5 kg of fat out of 10 kg of peripheral adipose tissue to the liver within a 3 year period , a realistic clinical situation.Therefore , in our opinion , adipose tissue is able to oxidise substantial amounts of fatty acids over a long period by mitochondrial beta oxidation . This process may over time attenuate ectopic deposition of those fatty acids . Since mitochondria also contribute to formation of glycerol 3-phosphate , needed for re-esterification of fatty acids within adipocytes , mitochondria in adipocytes have the ability to protect the organism to a certain extent against leakage of fatty acids into the circulation and thus , redistribution of body fat . As Frayn et al. -LSB- 5 -RSB- have pointed out , brown adipose tissue is indeed more active in uncoupled oxidative disposal of fatty acids compared with white tissue . Recent evidence also suggests that brown adipose tissue is present in variable amounts and at multiple sites in humans , contrary to earlier reports -LSB- 10 -RSB- . This recently recognised situation further enhances the capacity of the adipose compartment to remove fatty acids through uncoupled beta oxidation.In order to understand the mechanism by which mitochondria in adipocytes contribute to the control of body fat distribution during the development of the metabolic syndrome , additional studies are needed , comprising quantification of fatty acid fluxes in relation to re-esterification and oxidation in adipose tissue . These studies should include study of adipose tissue from individuals with normal glucose tolerance and from those with developing glucose intolerance .

O O O O O B O O O O O O O O O O O O O O B O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B I O B O O O O B I O O O O O O O O O O O O O O O B O O O O O O O O O O B I O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O B O B O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O B I O O B O O O O O O O O O O O O B I O B O B O B O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

adipocytes
fatty acids
haart
mitochondria


Management and prognostic factors of recurrent pleomorphic adenoma of the parotid gland : personal experience and review of the literature The aim of this study was to investigate the management and prognostic determinants of recurrent pleomorphic adenoma -LRB- RPA -RRB- . A retrospective analysis was performed to examine the clinical features , the prevalence of surgical complications , and new recurrences of RPA . Tumor recurrence rate was estimated by the Kaplan -- Meier method , and the prognostic value of some of the variables was tested by univariate analysis using the log rank test . The study focused on 33 patients , 18 female -LRB- 54.5 % -RRB- and 15 male -LRB- 45.5 % -RRB- , aged 12 -- 71 years -LRB- median 41 -RRB- . A total or extended total parotidectomy was performed in 16 cases -LRB- 48.5 % -RRB- , a superficial parotidectomy in 10 cases -LRB- 30.3 % -RRB- , and a local excision in 7 cases -LRB- 21.2 % -RRB- . In ten patients -LRB- 30.3 % -RRB- , a branch or the trunk of the facial nerve was deliberately sacrificed . Major complications included one unexpected definitive paralysis of the marginal mandibular branch of the facial nerve and 14 cases of Frey syndrome . Follow-up varied from 2 to 25 years -LRB- median 10.5 years -RRB- , and there were 11 new recurrences -LRB- 33.3 % -RRB- within a period varying from 1 to 16 years -LRB- median 6 years -RRB- . The estimated tumor recurrence rates were 14.1 ± 6.6 % at 5 years , 31.4 ± 9.4 % at 10 years , 43.0 ± 10.8 % at 15 years , and 57.2 ± 14.8 % at 20 years . Presence of a multinodular lesion and the type of intervention performed were significantly associated with a higher probability of recurrence . RPAs are prone to new recurrences , especially when multinodular and treated with a local excision . Surgical treatment should include facial nerve resection in selected cases . Follow-up for the patient 's lifetime is warranted . Introduction Pleomorphic adenoma is the most common neoplasm of the parotid gland . It is a benign tumor composed of epithelial and myoepithelial cells arranged in various morphological patterns . Thinning or absence of the pseudocapsule and the presence of fingerlike projections of the tumor have been observed in all histologic subtypes of pleomorphic adenoma , in particular the myxoid type -LSB- 1 -RSB- . Even though many hypotheses for recurrences of parotid gland pleomorphic adenoma have been advanced , including cell biological and genetic factors -LSB- 2 , 3 -RSB- , obvious or underestimated tumor spillage , incomplete excision , and violation of the pseudocapsule of the tumor are considered the only proven reasons contributing to recurrent disease -LSB- 4 -RSB- . This is why the abandonment of enucleation techniques in favor of more extended surgical procedures , which require the tumor to be excised with the surrounding normal tissue and the facial nerve to be identified and preserved , have dramatically reduced the recurrence rate of pleomorphic adenomas of the parotid gland from 20 -- 45 % to less than 4 % in the last decades -LSB- 5 -- 7 -RSB- . However , pure en bloc excision in parotid benign tumor disease has been considered impossible by most surgeons when the pleomorphic adenoma is lying on the facial nerve , and so partial enucleation is often the only surgical option in those cases -LSB- 5 , 7 -RSB- , thus explaining the possibility of recurrence even after proper surgery . Most of the recent literature on recurrent pleomorphic adenoma -LRB- RPA -RRB- of the parotid gland has attempted to analyze the causes of primary failure and discusses other possible risk factors for recurrence -LSB- 8 -- 12 -RSB- ; however , there are very few papers focused on the prognostic factors of RPA and retreatment policies -LSB- 13 -- 18 -RSB- . The present study was undertaken to address these important issues based on a follow-up period of up to 20 years . Here , we present our findings and review some of the more recent literature . Materials and methods A retrospective analysis of the charts of patients treated for benign parotid neoplasm between 1983 and 2004 was performed to identify those patients operated on for RPA . We collected information on patient demographics and clinical history , clinical features of the lesions , diagnostic work-up , the type of surgery , operative findings , facial nerve management , and adjuvant treatment . The study analyzed the prevalence of surgical complications and new recurrences . Statistical analysis was performed using the SPSS statistical package . Tumor recurrence rate was estimated by the Kaplan -- Meier method : the entry point was the date of surgery for a recurrent tumor seen for the first time in our department , and the end point was the date of a new recurrence or the date of last consultation for censored observations . The prognostic value of certain categorical variables -LRB- age , gender , number of previous operations , type of previous operation , lobe of origin , type of surgery performed for RPA , maximum diameter , facial nerve resection , presence of a pseudocapsule at the surgical margins , rupture of the pseudocapsule , number of neoplastic nodules , postoperative radiotherapy -RRB- was tested by univariate analysis using the log rank test . Results Among 524 patients treated for a benign parotid neoplasm between 1983 and 2004 , 33 underwent surgical treatment for RPA . Eighteen -LRB- 54.5 % -RRB- patients were female and 15 -LRB- 45.5 % -RRB- male ; their ages ranged from 12 to 71 years -LRB- median 41 -RRB- . None had been previously affected by a malignant neoplasm or submitted to radiation treatment . Seven patients were previously operated on in our department -LRB- with a prevalence of recurrence after first treatment of 1.3 % -RRB- and 26 patients had previous surgical treatment -LRB- s -RRB- performed elsewhere . A summary of the patient 's previous clinical history is reported in Table 1 . A swelling in the parotid region was the most significant complaint for 87.9 % -LRB- 29/33 -RRB- of patients when they came to our attention for the recurrence ; pain or facial nerve paresis was associated with the swelling in two cases . In four -LRB- 12.1 % -RRB- cases , the relapse was recognized by routine postoperative follow-up examinations . Other complaints , present from previous surgeries , were facial nerve paralysis in four patients and Frey syndrome in one . Table 1Summary of clinical history of patients treated for the first time at the Department of Otolaryngology of the University of Brescia for RPA of the parotid glandPrevious recurrences One63 .6 % -LRB- 21 -RRB- Two 18.2 % -LRB- 6 -RRB- Three 12.1 % -LRB- 4 -RRB- Four 6.1 % -LRB- 2 -RRB- Median time to recurrence First recurrence -LRB- 33 -RRB- 6 years -LRB- range 1 -- 23 -RRB- Second recurrence -LRB- 12 -RRB- 5 years -LRB- range 2 -- 28 -RRB- Third recurrence -LRB- 6 -RRB- 5.5 years -LRB- range 3 -- 19 -RRB- Fourth recurrence -LRB- 2 -RRB- 8 years -LRB- both patients -RRB- Last operation Subtotal parotidectomy 33.3 % -LRB- 11 -RRB- Local excision 33.3 % -LRB- 11 -RRB- Superficial parotidectomy 21.2 % -LRB- 7 -RRB- Not reported 12.1 % -LRB- 4 -RRB- Number of patients in parentheses Ultrasonography was the most frequently used diagnostic tool -LRB- 23 patients , 69.7 % -RRB- , followed by fine needle aspiration biopsy in 21 -LRB- 63.6 % -RRB- patients , magnetic resonance imaging in 14 -LRB- 42.4 % -RRB- , computed tomography in 3 -LRB- 9.1 % -RRB- , and sialography in 2 -LRB- 6.1 % -RRB- . The different types of diagnostic procedures employed reflect the multi-decade period analyzed . At present , after ultrasonography , which is routinely adopted for follow-up , magnetic resonance imaging is used to delineate the extension of the recurrent lesions . In 12 -LRB- 36.4 % -RRB- patients , the lesion involved the superficial lobe of the parotid gland , in 9 -LRB- 27.3 % -RRB- both lobes , and in 12 -LRB- 36.4 % -RRB- only the deep lobe was involved . In 5 -LRB- 15.2 % -RRB- patients the lesion also extended to the parapharyngeal space . Information on multinodularity of the lesion could be obtained for 31 patients : multiple lesions were observed in 24 -LRB- 77.4 % -RRB- of them . Surgical treatment was tailored to the single patient in an attempt to obtain a margin of healthy tissue -LRB- Table 2 -RRB- . Postoperative complications are listed in Table 3 . Nine -LRB- 27.3 % -RRB- patients were submitted to postoperative radiotherapy . There were no standard criteria for postoperative radiotherapy , but it was performed when the excision did not guarantee sufficient free margins of healthy tissue based on the judgment of the surgeon . Table 2Surgical treatment for RPA of the parotid gland at the Department of Otolaryngology of the University of BresciaTotal or extended total parotidectomy 48.5 % -LRB- 16 -RRB- Superficial parotidectomy 30.3 % -LRB- 10 -RRB- Local excision 21.2 % -LRB- 7 -RRB- Partial or total facial nerve resection30 .3 % -LRB- 10 -RRB- Number of patients in parenthesesTable 3Postoperative complications of patients treated for RPA of the parotid gland at the Department of Otolaryngology of the University of BresciaFrey syndrome 43.7 % -LRB- 14/32 -RRB- Temporary paresis of a branch or of the entire facial nerve30 .4 % -LRB- 7/23 -RRB- Definitive paralysis of the marginal mandibular branch of the facial nerve4 .3 % -LRB- 1/23 -RRB- Salivary fistula3 .0 % -LRB- 1/33 -RRB- Keloid3 .0 % -LRB- 1/33 -RRB- The patients were followed by yearly ultrasonography . Follow-up varied from 2 to 25 years -LRB- median 10.5 years -RRB- . Three patients died from a metachronous malignant neoplasm , one of them after a new recurrence . The other patients are still alive . Overall , 11 -LRB- 33.3 % -RRB- new recurrences were observed . No patient with a single node had a recurrence . Four of the new recurrences had been operated on again and one had a further recurrence . Seven patients with small -LRB- less than 1 cm -RRB- and stable new recurrences are still on follow-up . The time for new recurrence varied from 1 to 16 years -LRB- median 6 years -RRB- . There were 2 cases of focal atypical cells but no malignant transformation in the new recurrences . Distribution of the recurrences in relation to the different variables under analysis is shown in Table 4 . The estimated tumor recurrence rates were 14.1 ± 6.6 % at 5 years , 31.4 ± 9.4 % at 10 years , 43.0 ± 10.8 % at 15 years , and 57.2 ± 14.8 % at 20 years -LRB- Fig. 1 -RRB- . Among the variables evaluated with the log-rank test , only the presence of a multinodular recurrence and the type of intervention performed were significantly associated with a higher probability of a new recurrence -LRB- Table 5 -RRB- . The high variation in tumor recurrence rates observed after 20 years of follow-up -LRB- Table 5 -RRB- is probably related to the low number of patients with longer follow-up times . Table 4Distribution of the new recurrences after treatment for RPA of the parotid gland at the Department of Otolaryngology of the University of Brescia -LRB- No . = 11 ; 33.3 % -RRB- Variable % P-valueAge ≤ 30 years -LRB- 3/10 -RRB- 30.01 > 30 years -LRB- 8/23 -RRB- 34.8 GenderFemale -LRB- 4/18 -RRB- 22.20.1 Male -LRB- 7/15 -RRB- 46.7 Number of previous operations1 -LRB- 7/21 -RRB- 33.31 > 1 -LRB- 4/12 -RRB- 33.3 Type of previous operationsEnucleation -LRB- 2/11 -RRB- 18.20.2 Parotidectomy -LRB- 8/18 -RRB- 44.4 Lobe of originSuperficial -LRB- 5/21 -RRB- 23.80.1 Deep -LRB- 6/12 -RRB- 50.0 Intervention performedParotidectomy -LRB- 6/26 -RRB- 23.10.02 Other -LRB- 5/7 -RRB- 71.4 Maximum diameter ≤ 2 cm -LRB- 7/16 -RRB- 43.80.3 > 2 cm -LRB- 3/15 -RRB- 20.0 Facial nerve resectionNo -LRB- 7/23 -RRB- 30.40.7 Yes -LRB- 4/10 -RRB- 40.0 Involvement of surgical marginsNo -LRB- 8/23 -RRB- 34.80.5 Yes -LRB- 2/8 -RRB- 25.0 Capsular ruptureNo -LRB- 6/23 -RRB- 26.10.2 Yes -LRB- 4/8 -RRB- 50.0 Multiple nodulesNo -LRB- 0/7 -RRB- 00.04 Yes -LRB- 10/24 -RRB- 41.7 Postoperative radiotherapyNo -LRB- 9/24 -RRB- 37.50.3 Yes -LRB- 2/9 -RRB- 22.2 Fig. 1Estimated recurrence rate after treatment for recurrent pleomorphic adenoma of the parotid gland at the Department of Otolaryngology of the University of Brescia -LRB- Kaplan -- Meier method -RRB- Table 5Estimated tumor recurrence rates after treatment for RPA of the parotid gland at the Department of Otolaryngology of the University of Brescia -LRB- log-rank test -RRB- VariableEstimated tumor recurrence rates -LRB- % -RRB- P-value5-year10-year15-year20-yearAge ≤ 30 years -LRB- 10 -RRB- 12.5 ± 11.741.7 ± 18.6 -- -- 0.8 > 30 years -LRB- 23 -RRB- 14.7 ± 7.926.9 ± 10.542.4 ± 12.856.8 ± 15.7 GenderFemale -LRB- 18 -RRB- 13.4 ± 9.030.7 ± 13.130.7 ± 13.130.7 ± 13.10.4 Male -LRB- 15 -RRB- 14.3 ± 9.430.7 ± 13.056.7 ± 15.171.1 ± 16.3 Number of previous operations1 -LRB- 21 -RRB- 11.8 ± 7.925.4 ± 11.145.6 ± 14.763.7 ± 17.80.9 > 1 -LRB- 12 -RRB- 18.5 ± 11.941.8 ± 16.341.8 ± 16.341.8 ± 16.3 Type of last previous operationEnucleation -LRB- 11 -RRB- 9.1 ± 8.724.2 ± 15.624.2 ± 15.624.2 ± 15.60.2 Parotidectomy -LRB- 18 -RRB- 20.0 ± 10.345.1 ± 14.058.9 ± 15.9100 Lobe of originSuperficial -LRB- 21 -RRB- 013.8 ± 9.132.1 ± 13.654.8 ± 20.60.07 Deep -LRB- 12 -RRB- 35.8 ± 14.459.9 ± 16.459.9 ± 16.459.9 ± 16.4 Intervention performedParotidectomy -LRB- 26 -RRB- 5.3 ± 5.116.4 ± 8.730.5 ± 11.747.9 ± 17.40.001 Other -LRB- 7 -RRB- 42.9 ± 18.7 -- -- -- Maximum diameter ≤ 2 cm -LRB- 16 -RRB- 19.6 ± 10.226.9 ± 11.644.3 ± 14.072.2 ± 20.90.4 > 2 cm -LRB- 15 -RRB- 10.0 ± 9.532.5 ± 15.532.5 ± 15.532.5 ± 15.5 Facial nerve resectionNo -LRB- 23 -RRB- 9.4 ± 6.327.2 ± 10.635.3 ± 12.151.4 ± 16.70.4 Yes -LRB- 10 -RRB- 25.0 ± 15.340.0 ± 18.2 -- -- Involvement of surgical marginsNo -LRB- 23 -RRB- 14.0 ± 7.524.7 ± 9.737.9 ± 11.753.4 ± 16.00.3 Yes -LRB- 8 -RRB- 25.0 ± 21.762.5 ± 28.6 -- -- Capsular ruptureNo -LRB- 23 -RRB- 16.4 ± 8.838.7 ± 12.838.7 ± 12.838.7 ± 12.80.8 Yes -LRB- 8 -RRB- 12.5 ± 11.712.5 ± 11.740.0 ± 18.2100 Multiple nodulesNo -LRB- 7 -RRB- 00000.02 Yes -LRB- 24 -RRB- 19.6 ± 8.936.8 ± 11.253.2 ± 13.4100 Postoperative radiotherapyNo -LRB- 24 -RRB- 14.2 ± 7.734.3 ± 11.848.9 ± 12.965.9 ± 16.40.3 Yes -LRB- 9 -RRB- 12.5 ± 11.625.0 ± 15.325.0 ± 15.325.0 ± 15.3 Discussion It is generally accepted that the standard treatment of pleomorphic adenoma of the parotid gland is a formal parotidectomy or at least the removal of the tumor with a surrounding cuff of normal parotid tissue after identification , dissection and preservation of the facial nerve -LSB- 7 -RSB- . Unfortunately , if the tumor is in contact with the facial nerve , even a total parotidectomy can not guarantee the presence of a cuff of normal tissue , because the surgeon must carry on the dissection between the tumor and the branches of the nerve with the risk of leaving microscopic disease behind or causing microscopic spillage of the tumor . This accounts for 1 -- 4 % probability of recurrence -LSB- 5 , 7 -RSB- . These low percentages , together with the increasing number of institutions that treat parotid neoplasms , have reduced the number of cases of RPA being referred to a single institution ; this , in turn , has contributed to a lack of experience in managing these tumors . Moreover , with the number of reported patients usually varying from less than 15 to about 50 -LSB- 6 , 8 , 9 , 11 , 12 , 14 -- 17 , 19 -- 24 -RSB- , and only a few studies reporting about a hundred patients -LSB- 10 , 13 , 18 , 25 -RSB- , there has been relatively little discussion of the prognostic factors for RPA -LSB- 13 -- 18 -RSB- . Follow-up of parotidectomies is currently performed by ultrasonography , but most patients do not avail themselves of regular follow-up and only 12.1 % of our patients with an RPA were diagnosed by routine ultrasonography , while the majority came to our observation for a swelling in the parotid region . In 77.4 % of our patients , there was a multinodular lesion -LRB- in Stennert et al. -LSB- 4 -RSB- , 90 % were multinodular at pathologic examination -RRB- , reinforcing the pathogenetic hypothesis of incomplete excision , violation of the pseudocapsule of the tumor , or obvious or underestimated tumor spillage during the first surgery due to the impossibility of obtaining healthy free margins when the lesion is adjacent to the facial nerve . Delineation of the extension of the RPA is better depicted by MRI , which better demonstrates the multinodular nature of the disease -LSB- 24 -RSB- and potential deep-lobe or parapharyngeal extension . Many patients that we treated came from other institutions without clear documentation of their previous treatment -LRB- s -RRB- ; in such cases , MRI can also help in determining the amount of parotid gland still present after any previous surgery . The signal and enhancement characteristics of RPA of the parotid gland are nonspecific -LSB- 26 -RSB- . The lesions are usually round ; they are of low intensity on T1-weighted images and of high intensity on T2-weighted MR images . With contrast administration , the lesions show mild enhancement -LSB- 26 -RSB- . However , MRI can also be inadequate for identifying all nodules , and frequently the surgeon 's microscope or the pathologist 's microscope reveals many more nodules than suspected from clinical assessment -LSB- 4 -RSB- . Management options for RPA include observation only , localized resection , resection with facial nerve dissection , wide-field resection with facial nerve sacrifice , and radiotherapy -LSB- 2 , 4 , 24 -RSB- . Only observation has been suggested for the elderly or medically infirm patient -LSB- 2 , 4 , 24 -RSB- . When surgical excision is considered , it should be tailored to the individual patient -LSB- 19 , 23 -RSB- . In general , it is recommended to resect the scars of any previous surgery . Localized resection of the tumor has been employed after multiple recurrences or after previous total parotidectomy when it is the only option to preserve the facial nerve -LSB- 10 , 14 -RSB- . Superficial or total parotidectomy , depending on the location of the recurrence , has been suggested when the previous operation was a simple local excision -LSB- 6 , 8 -- 15 , 17 , 18 , 20 , 23 -- 25 -RSB- . Radical and extended parotidectomies have been considered for patients with infiltration of branches or the main trunk of the facial nerve , and in cases of multinodular recurrences -LSB- 9 , 11 -- 13 , 15 , 18 , 23 -- 25 -RSB- . In particular , facial nerve resection has been suggested for patients with a history of multiple recurrences or failed radiotherapy -LSB- 24 -RSB- . In 30.3 % of our patients , the resection of at least a branch of the facial nerve was performed -LRB- Table 2 -RRB- . Facial nerve dissection and preservation were performed in all cases with still healthy parotid tissue between the nerve and the tumor , and in the case of facial nerve encasement when a patient refused facial nerve resection . The observation of many cases with unexpected multinodularity persuaded Stennert et al. -LSB- 4 -RSB- to routinely perform a total parotidectomy for all RPAs . However , such an aggressive policy of treatment does not prevent leaving microscopic residuals ; indeed , a recent study by the same group reported a 52 % probability of new recurrences -LSB- 18 -RSB- . The role of radiotherapy remains questionable -LSB- 9 , 13 , 16 , 17 , 24 , 27 -RSB- . Some authors suggest that radiotherapy should be reserved only for malignant tumors since it carries , especially in young people , a risk of malignant transformation that increases over time -LSB- 17 -RSB- . Nonetheless , its use is not infrequent -LSB- 6 , 9 , 13 -- 16 , 19 , 20 , 27 -RSB- . Jackson et al. -LSB- 9 -RSB- used postoperative radiotherapy in patients in whom histology showed that resection margins of the specimen were not free of tumor or in those with intraoperative spillage of tumor . Renehan et al. -LSB- 13 -RSB- recommended postoperative radiotherapy in multinodular recurrences . Douglas et al. -LSB- 16 -RSB- reported their experience of fast neutron radiotherapy and recommended radiotherapy for selected cases : when complete extirpation is not possible , when sacrifice of the facial nerve is necessary , or after multiple recurrences . Glas et al. -LSB- 17 -RSB- suggested postoperative radiotherapy for difficult cases in which further surgical treatment of recurrent disease is not recommended for technical reasons . Kamida et al. -LSB- 27 -RSB- suggested the use of stereotactic radiosurgery for difficult selected cases , such as the patient they treated for skull base invasion with no regrowth after 5 years . Our experience , including only nine patients , is too limited to draw any conclusion about indications for radiation therapy . Complications of treatment of RPA are similar to those for primary surgery for parotid pleomorphic adenoma . The main difference is the higher risk of facial nerve damage in the former , with unexpected permanent facial nerve paralysis in more than 30 % of cases -LSB- 24 -RSB- . Even though we did not have cases of malignant transformation , a high incidence is expected , especially after multiple recurrences , reaching up to 15 % in the experience of Mercante et al. -LSB- 22 -RSB- . The main purpose of our study was to analyze risk factors for new recurrence after treatment for RPA . The percentage of new recurrences varies from 10 to 58 % -LSB- 8 , 10 , 12 -- 15 , 17 -- 20 , 23 -- 25 -RSB- . In two surveys of the literature with a 10-year follow-up , the prevalence of a new recurrence was 43 and 45 % , respectively -LSB- 12 , 25 -RSB- . Wittekindt et al. -LSB- 18 -RSB- reported a recurrence rate increasing from 42 % at 5 years to 75 % at 15 years . We observed a prevalence of 33.3 % with an estimation of recurrence rate increasing from 14.1 ± 6.6 % at 5 years to 31.4 ± 9.4 % at 10 years , 43.0 ± 10.8 % at 15 years , and 57.2 ± 14.8 % at 20 years -LRB- Fig. 1 -RRB- . Among the variables tested with the log-rank test against the estimated new recurrence rate -LRB- Table 5 -RRB- , just two were significantly associated with the likelihood of developing a new recurrence : local excision of the RPA instead of a formal parotidectomy and the presence of multinodular disease . To date , no new recurrences have been observed for uninodular recurrences . There was a trend toward increased risk of a new recurrence for deep-lobe localization , although it was not statistically significant . Few papers have analyzed prognostic factors for RPA -LSB- 13 -- 18 -RSB- . Renehan et al. -LSB- 13 -RSB- studied 114 patients with first-recurrence RPA and reported significantly better , new recurrence-free survival for patients treated with postoperative radiotherapy when the RPA was multinodular . Age , gender , time to first recurrence , and the type of prior treatment were all not significant predisposing factors for a new recurrence -LSB- 13 -RSB- . Yugueros et al. -LSB- 14 -RSB- followed 39 patients treated for RPA at their institution : they found no significant difference in development of another recurrence in relation to gender , time after the previous treatment , size of the tumor , type of resection , and postoperative radiotherapy . Carew et al. -LSB- 15 -RSB- analyzed 31 patients treated for RPA and concluded that the extent of the first parotid operation was the only factor with a statistically significant impact on tumor control after resection of the recurrence . Local control was achieved in all patients whose initial procedures involved local excision . Superficial lobe location and postoperative radiotherapy showed better results but were not significant -LSB- 15 -RSB- . Fast neutron radiotherapy of 16 patients with residuals of RPA stopped progression of the tumor in patients without gross residuals , without multifocal disease and with an interval from diagnosis of less than 25 years ; however , the results were not statistically significant -LSB- 16 -RSB- . Glas et al. -LSB- 17 -RSB- analyzed 52 patients with RPA , treated after one or more recurrences : no significant influence was seen in the recurrence rate with respect to gender , age at initial treatment , time of first recurrence , and intraoperative tumor spillage . Wittekindt et al. -LSB- 18 -RSB- , in a group of 108 patients , observed a significantly higher rate of new recurrences in female patients , in younger patients , and in patients treated with a simple enucleation . Conclusion The management of RPA is a major challenge for the clinician . RPAs , particularly multinodular tumors , are prone to new recurrences especially when treatment of the initial tumor is performed according to currently accepted standards . MRI is considered to be the best tool to delineate the extension of the lesion , although it can miss microscopic nodules . Options for management include pure observation , not only for the elderly or infirm , but also in cases of small lesions until they begin to grow . When surgery is elected , it should be tailored to the single patient , because even if on one side a limited local excision is considered acceptable , on the other side a total or extended parotidectomy may be inadequate to control an RPA adjacent to the nerve . In these cases , facial nerve resection and reconstruction must also be considered and discussed with the patient in the preoperative counseling . Postoperative radiotherapy is an option , particularly for older patients for whom the risk of inducing other malignancies is considered to be low . Follow-up for patients treated for RPA should be done with regular ultrasonography for the lifetime of the patient and with magnetic resonance in selected cases with deep lobe or parapharyngeal involvement . All patients should be informed about the possibility of the need for multiple treatment procedures .

O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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