Insulin-regulated aminopeptidase in adipocyte is Cys-specific and affected by obesity

in Journal of Molecular Endocrinology

Insulin-regulated aminopeptidase (IRAP, EC 3.4.11.3) in adipocytes is well known to traffic between high (HDM) and low (LDM) density microsomal fractions toward the plasma membrane (MF) under stimulation by insulin. However, its catalytic preference for aminoacyl substrates with N-terminal Leu or Cys is controversial. Furthermore, possible changes in its traffic under metabolic challenges are unknown. The present study investigated the catalytic activity attributable to EC 3.4.11.3 in HDM, LDM and MF from isolated adipocytes of healthy (C), food deprived (FD) and monosodium glutamate (MSG) obese rats on aminoacyl substrates with N-terminal Cys or Leu, in absence or presence of insulin. Efficacy and reproducibility of subcellular adipocyte fractionation procedure were demonstrated. Comparison among HDM vs LDM vs MF intragroup revealed that hydrolytic activity trafficking from LDM to MF under influence of insulin in C, MSG and FD is only on N-terminal Cys. In MSG the same pattern of anterograde traffic and aminoacyl preference occurred independently of insulin stimulation. The pathophysiological significance of IRAP in adipocytes seems to be linked to comprehensive energy metabolism related roles of endogenous substrates with N-terminal cysteine pair such as vasopressin and oxytocin.

Abstract

Insulin-regulated aminopeptidase (IRAP, EC 3.4.11.3) in adipocytes is well known to traffic between high (HDM) and low (LDM) density microsomal fractions toward the plasma membrane (MF) under stimulation by insulin. However, its catalytic preference for aminoacyl substrates with N-terminal Leu or Cys is controversial. Furthermore, possible changes in its traffic under metabolic challenges are unknown. The present study investigated the catalytic activity attributable to EC 3.4.11.3 in HDM, LDM and MF from isolated adipocytes of healthy (C), food deprived (FD) and monosodium glutamate (MSG) obese rats on aminoacyl substrates with N-terminal Cys or Leu, in absence or presence of insulin. Efficacy and reproducibility of subcellular adipocyte fractionation procedure were demonstrated. Comparison among HDM vs LDM vs MF intragroup revealed that hydrolytic activity trafficking from LDM to MF under influence of insulin in C, MSG and FD is only on N-terminal Cys. In MSG the same pattern of anterograde traffic and aminoacyl preference occurred independently of insulin stimulation. The pathophysiological significance of IRAP in adipocytes seems to be linked to comprehensive energy metabolism related roles of endogenous substrates with N-terminal cysteine pair such as vasopressin and oxytocin.

Introduction

During the prandial period, insulin stimulates the translocation of an intracellular vesicle from low density microsomal (LDM) fraction to the plasma membrane (MF) of adipocytes (Watson & Pessin 2001, Zeyda & Stulnig 2009, Jordens et al. 2010, Dimitriadis et al. 2011). This vesicle contains glucose transporter type 4 (GLUT4) and insulin-regulated aminopeptidase (IRAP, EC 3.4.11.3) (Keller et al. 1995, Keller 2004, Jordens et al. 2010). Hydrolytic activity on angiotensin (Ang)-3 (Ruster & Wolf 2013), lysil-bradykinin (Ino et al. 2003), vasopressin (AVP) (Ino et al. 2003) and oxytocin (OXT) (Keller 2004), as well Ang-4 receptor function (Albiston et al. 2010) have been attributed to EC 3.4.11.3. Several studies show variation of EC 3.4.11.3 protein expression in LDM, MF and also in compartment of cellular protein origin, corresponding to high density microsomal (HDM) fraction (Alberts et al. 2008), under different pathophysiological conditions (Maianu et al. 2001, Jordens et al. 2010). LDM consists of microsomes without ribosomes and with portions of endoplasmatic reticulum (ER) and fragments of Golgi apparatus. HDM consists of microsomes with ribosomes adhered to its outer surface. The inner side of microsomes is biochemically equivalent to the lumen of the ER (Alberts et al. 2008). However, concomitant hydrolytic activity of EC 3.4.11.3 has been rarely evaluated in these locations (Sano et al. 2005). In general, catalytic ability has been assessed only under basal conditions and using aminoacyl substrates with N-terminal Leu (Ross et al. 1996, Sano et al. 2005). Curiously, hydrolytic activity in aminoacyl substrates with N-terminal Cys has been also consensually attributed to IRAP based on rare experimental reports (Carrera-González et al. 2011). This consensus was experimentally reinforced by recent findings that hydrolytic activity of MF from adipocytes in aminoacyl substrate with N-terminal Cys prevails over that with N-terminal Leu (R F Alponti and P F Silveira, unpublished observations). On the other hand, disturbances related with insulin are critical in the development of various diseases (Gross et al. 2004, Tesauro & Cardillo 2011). For example, insulin resistance in muscle and adipose tissues is a very common occurrence in obese (Bonala et al. 2013, Lo et al. 2013).

This study aimed to identify whether intracellular traffic regulated by insulin in adipocytes from healthy (C), food deprived (FD), and monosodium glutamate (MSG) obese rats is preferentially attributable to EC 3.4.11.3 hydrolytic activity on N-terminal Cys or Leu.

Experimental procedures

Animals and treatments

Immediately after birth, male Wistar rats were housed with a lactant female in a polypropylene box (inside length×width×height=56 cm×35 cm×19 cm) within a ventilated container (Alesco Ind. Com Ltda, Monte Mor, SP, Brazil) under controlled temperature (24±2 °C), relative humidity (65±1%) and 12 h light:12 h darkness photoperiod (lights on at 0600 h). Twenty-four hours after birth, the animals were subdivided into two groups, treated according to the methodology described by Alponti & Silveira (2010). The i-MSG group received a daily s.c. bolus injection of l-glutamic acid monosodium salt (Sigma) in saline 0.9% (4 mg/g body mass), in the cervical region, between 0730 and 0900 h of light period (1330–1500 h), at a maximum volume of 0.2 ml, until they were 10 days old. The i-control group received the same volume of saline 0.9% s.c., in the same scheme as previously described. On the 22nd day, the animals were weaned and the female was removed from the cage. On the 90th day, obesity was evaluated by Lee index, calculated by body mass (g)0.33/naso-anal length (cm) (Nakagawa et al. 2000, Kaufhold et al. 2002). Thus, obese (MSG) animals were selected from i-MSG group by Lee index >0.300 and the i-control rats were subdivided into two groups: control (Lee index ≤0.300) and FD control (control animals fasted for 72 h). Food deprivation was performed by transferring pairs of animals into metabolic cages without food for 72 h. Drinking water ad libitum was available all the time.

The conducts and procedures involving animal experiments were approved by the Butantan Institute Committee for Ethics in Animal Experiments (License number CEUAIB 684/2009) in compliance with the recommendations of the National Council for the Control of Animal Experimentation of Brazil (CONCEA). All efforts were made to minimize suffering.

Adipocytes isolation

After euthanasia by decapitation, the retroperitoneal fat pad was removed through manual dissection and washing with saline 0.9%. Total mass (g) of fat pad was measured, 3 g was separated and submitted to collagenase digestion as described by Rodbell (1964). Briefly, this quantity of fat pad was added to 9 ml of DMEM (Cultilab, Campinas, SP, Brazil) containing 25 mM HEPES (pH 7.5), 4% BSA (Sigma) and 45 mg of collagenase (Sigma), and then incubated at 37 °C, for 1 h, under gentle shaking. Subsequently, this mixture was washed with eight volumes of buffered washing solution (115 mM NaCl, 0.8 mM MgSO4.7H2O, 5.3 mM KCl, 1.4 mM CaCl2.2H2O, 0.89 mM NaH2PO4.H2O, 25 mM HEPES, 1 mM Na pyruvate, 145 mM BSA; pH 7.4, at 25 °C), and then filtered through a nylon mesh. This filtrate was centrifuged at 13 g for 1 min, at 25 °C. The pellet containing vascular stroma (capillaries, endothelial cells, mast cells, macrophages and epithelial cells) was removed by suction and discarded, while the supernatant, containing the suspension of adipocytes, was washed and centrifuged again at the same conditions three more times. The resultant suspension of isolated adipocytes was microscopically observed to verify the absence of vascular stroma.

Incubation of isolated adipocytes with insulin

KRBH-BSA buffer (121 mM NaCl, 1.2 mM MgSO4, 4.9 mM KCl, 2.4 mM NaH2PO4, 0.33 mM CaCl2, 20 mM HEPES, pH 7.4, 4% BSA, 5 mM glucose) was added to adipocytes suspension obtained from each animal, at a ratio of 3 ml of buffer for each gram of adipose tissue that originated the suspension. Subsequently, saline or 10−6 M insulin (Sigma) at a maximum volume of 60 μl were added and then the mixture was incubated for 15 min at 37 °C under gentle agitation. Insulin was dissolved in water and diluted in KRBH-BSA buffer. After the incubation period, the mixture was immediately used in the next procedure.

Obtaining HDM, LDM and MF

Based on McKeel & Jarett (1970), the suspension of adipocytes isolated from 3 g of fat pad from each animal was put in 2.3 ml homogenization buffer (20 mM Tris–HCl/255 mM sucrose, pH 7.4, 20 °C) (HB) and sonicated for 20 s at amplitude of 20 μm at 20 kHz. The resulting suspension was then centrifuged at 2000 g, for 10 min and the top layer containing solidified fat cake was discarded, and the infranatant was collected. Subsequently, 0.1% (relative to final volume) Triton X-100 were added to this infranatant, which was homogenized in a potter glass at 800 r.p.m., for 3 min and centrifuged at 2000 g, for 10 min. The new top layer of remaining fat was discarded, and the infranatant was collected. Afterward 2.3 ml HB was added to this infranatant, which was homogenized by ten strokes with the teflon pestle in a potter glass and then ultracentrifuged (Hitachi model HIMAC CP60E) (16 000 g for 15 min). Again, the new top layer formed was carefully removed and discarded. The intermediate layer (CI-1) was removed and reserved for subsequent preparation of high and LDM fractions. The pellet (P-1) was resuspended in 2.3 ml HB and ultracentrifuged again at the same conditions (16 000 g for 15 min). The supernatant was discarded, the pellet (P-2) was resuspended in 2 ml HB and carefully transferred to another tube on a layer of sucrose solution (1.12 M sucrose containing 20 mM Tris–HCl previously applied to the bottom of the tube) and, thus, ultracentrifuged (101 000 g for 70 min). The intermediated layer (CI-2) formed was collected and resuspended in 1.9 ml HB and ultracentrifuged (48 000 g for 45 min). After this ultracentrifugation, the pellet (P-3) formed was resuspended in 1.9 ml HB and ultracentrifuged again (48 000 g for 45 min). The new pellet (P-4) containing plasma MF was solubilized in 1.9 ml HB containing 0.1% Triton X-100. Previously reserved CI-1 was ultracentrifuged (48 000 g for 20 min). The pellet (P-5) obtained was reserved for preparation of HDM fraction and the supernatant containing LDM fraction was ultracentrifuged (180 000 g for 70 min). The pellet formed (P-6) from this last ultracentrifugation was resuspended in 2.5 ml HB and ultracentrifuged again at the same conditions. The new pellet thus obtained contains LDM fraction (LDM) and was resuspended in the same volume with HB containing 0.1% Triton X-100. Previously reserved P-5 was resuspended in 2.5 ml HB and ultracentrifuged (48 000 g for 20 min). The new pellet thus obtained contains HDM fraction and was resuspended in the same volume (2.5 ml) with HB containing 0.1% Triton X-100. All procedures were carried out at 4 °C. Plasma MF, and high and LDM fractions were immediately frozen on dry ice and stored at −80 °C until their use.

Protein

Protein content (HDM, LDM and MF) was measured in triplicate at 630 nm in a Bio-Tek FL600FA microplate fluorescence/absorbance reader (BioTek, Winooski, VT, USA) by Bradford method (Bradford 1976) with a Bio-Rad protein assay kit (Bio-Rad) using BSA dissolved in the same sample diluent as standard.

Two-dimensional gel electrophoresis

Protein samples (80 μg) from HDM, LDM and FM obtained from the same animal were first individually precipitated with acetone (Jiang et al. 2004). According to the methodology described by Luna et al. (2013), each pellet thus obtained was suspended in 125 μl of DeStreak rehydration solution and 0.5% immobilized pH gradient (IPG) buffer pH 3–10 (GE Healthcare, Uppsala, Sweden). IPG strips of 7 cm, pH range 3–10, were passively rehydrated overnight at room temperature. Isoelectric focusing (IEF) was performed on an Ettan IPGphor II (GE Healthcare) using a program for a total of 5952 Vh according to the manufacturer's suggestion, including an initial step at low voltage of 100 V for 2 h to allow salt to migrate out of the strip. After IEF, the strips were initially incubated with equilibration buffer (75 mM Tris–HCl, pH 8.8, 6 M urea, 30% glycerol, 2% SDS, 0.002% bromophenol blue) containing 1% dithiothreitol (DTT) for 15 min. Next, solutions were discarded and the strips were incubated in an equilibration buffer containing 2.5% iodoacetamide for 15 min at room temperature. After equilibration, the strips were applied to the second dimension to separate proteins on a 12.5% SDS–PAGE and run on an Mini-PROTEAN Tetra electrophoresis system (Bio-Rad) according to the manufacturer's instructions. After running the gels, they were fixed (5% acetic acid, 20% methanol) for at least 30 min and stained with Coomassie Brilliant Blue G for 3 days. The stained gels were scanned on ImageScanner III (GE Healthcare). The identity and densitometry of the well-stained spots were analyzed in digitized images (8-bit grayscale, 300 dpi) by using the ImageMaster 2D Platinum v7.0 Software (GE Healthcare). Biological samples of three healthy animals were analyzed.

Peptidase activities

Cystyl-aminopeptidase (CysAP) and leucyl-aminopeptidase (LeuAP) activities were quantified based on the amount of β-naphthylamine released (Gasparello-Clemente et al. 2003) as the result of incubation (180 min, 37 °C), in 96-well flat-bottom microplates, of adipocyte fractions (HDM, LDM, MF), in triplicate, with prewarmed solution of 1.5 mM l-Cys-di-β-naphthylamide (Sigma) (solubilized in 0.012 N HCl) in Tris maleate buffer 0.05 M, pH 6.0, with BSA 0.1 mg/ml (CysAP activity) or 1.5 mM Leu-β-naphthylamide (Sigma) (solubilized in methanol) in phosphate buffer 0.05 M, pH 7.4, with BSA 0.1 mg/ml, 1 mM DL-DTT (LeuAP activity). The β-naphthylamine content was estimated fluorometrically (with a Bio-Tek FL600FA microplate fluorescence reader) at 415 nm emission wavelength and 335 nm excitation wavelength. The fluorescence value obtained at zero time (blank) was subtracted, and the relative fluorescence was then converted to picomoles of β-naphthylamine by comparison with the standard curve of β-naphthylamine (Sigma) dissolved in the same diluent used in the incubation. CysAP and LeuAP activities were expressed as picomoles of hydrolyzed substrate per minute per milligram of protein. The existence of a linear relationship between hydrolysis time and protein content in the fluorometric assay was a prerequisite.

Data analysis and results presentation

The quantitative data were shown as mean±s.e.m. and statistically analyzed using the software GraphPad Prism (GraphPad, La Jolla, CA, USA). Regression analysis was performed in order to obtain standard curves of protein and β-naphthylamine. ANOVA, followed by multiple comparison Tukey test, when differences were detected, was used to compare the values among three subcellular fractions. Unpaired two-side Student's t-test were used to compare values between two subcellular fractions. In all the calculations, a minimum critical level of P<0.05 was set.

Results

Simple visual inspection of the gels evidenced similar profiles in the same fraction among the different animals. Representative gel images of these profiles (Fig. 1) show that well-stained protein spots had molecular mass (MM) and isoelectric point (pI) respectively ranging between 34–225 kDa and 4.96–7.79 for HDM; 34–225 kDa and 3.56–8.00 for LDM; and 37–92 kDa and 4.96–8.00 for MF. HDM presented four spots (C, D, E and F) shared with LDM, being only the spot C with lower density than in LDM (Table 1). In addition to these four spots shared with HDM, LDM presented one peculiar (H) and three others spots shared with MF (I, J and K), two (I and K) with higher density than in MF (Table 1). MF presented one peculiar spot (L). The spots A, B and G were present in HDM, LDM and MF, being A and B with higher density in LDM than in HDM and MF (Table 1).

Figure 1
Figure 1

Representative 2-DE gel images of (i) high (HDM) and (ii) low (LDM) density microsomal and (iii) plasma membrane (MF) fractions from isolated adipocytes of three healthy animals. Samples (80 μg) were applied to 3–10 IPG strips followed by electrophoresis on 12.5% acrylamide gels. Spots are designated A to L.

Citation: Journal of Molecular Endocrinology 55, 1; 10.1530/JME-14-0321

Table 1

Comparison of well-stained protein spots in two-dimensional gel electrophoresis (2-DE) images. Values are means±s.e.m. of three healthy animals

SPOTM.M (kDa)pIDensity
HDMLDMFM
A394.960.078±0.013a0.212±0.015b0.064±0.011a
B565.310.073±0.013a0.195±0.029b0.066±0.001a
C725.810.354±0.0730.836±0.096*none
D1435.830.093±0.0100.152±0.046none
E2255.820.104±0.0410.323±0.163none
F347.480.101±0.0060.069±0.019none
G377.790.090±0.0220.144±0.0410.049±0.001
H743.56none0.210±0,071none
I447.31none0.143±0.0240.041±0.011*
J467.42none0.175±0.0630.089±0.005
K458none0.242±0.0630.049±0.002*
L925.84nonenone0.147±0.080

None, absent spot; M.M, molecular mass; pI, isoelectric point; HDM, high density microsomal; LDM, low density microsomal; MF, plasma membrane. HDM vs LDM vs MF (ANOVA, P<0.05; multiple comparison Tukey test, P<0.05, different letters (a,b) indicate significant differences of spot density among subcellular fractions). When one spot was absent in any fraction, the two others fractions were compared using the unpaired two-side Student's t-test (*P<0.05).

Figure 2 shows no differences of LeuAP activity among HDM, LDM and from C, FD and MSG under basal or insulin-stimulated conditions.

Figure 2
Figure 2

Leucyl-aminopeptidase (LeuAP) activity of high (HDM) and low (LDM) density microsomal and plasma membrane (MF) fractions from isolated adipocytes of retroperitoneal fat pad of control (C), food deprived (FD) and obese (MSG) rats in basal condition or under insulin stimulation. Values are mean±s.e.m. from triplicates of n animals (n in parenthesis over the bars). ANOVA, HDM vs LDM vs MF: basal (C, P=0.5125; FD, P=0.7072; MSG, P=0.6882); insulin (C, P=0.8962; FD, P=0.2894; MSG, P=0.2511).

Citation: Journal of Molecular Endocrinology 55, 1; 10.1530/JME-14-0321

Figure 3 shows no differences of CysAP activity among HDM, LDM and MF from C and FD under basal condition. However, under basal condition this activity in MSG was higher in MF than in HDM and LDM. After insulin stimulation, CysAP activity in MSG remained higher in MF and became also higher in MF than in HDM and LDM in C and FD.

Figure 3
Figure 3

Cystyl-aminopeptidase (CysAP) activity of high (HDM) and low (LDM) density microsomal and plasma membrane (MF) fractions from isolated adipocytes of retroperitoneal fat pad of control (C), food deprived (FD) and obese (MSG) rats in basal condition or under insulin stimulation. Values are mean±s.e.m. from triplicates of n animals (n in parenthesis over the bars). ANOVA, HDM vs LDM vs MF: basal (C, P=0.3973; FD, P=0.8675; MSG, P=0.0095); insulin (C, P=0.0031; FD, P=0.0049; MSG, P=0.0118. Multiple comparison Tukey test (P<0.05, different letters in the same group and stimulus indicate significant differences).

Citation: Journal of Molecular Endocrinology 55, 1; 10.1530/JME-14-0321

Discussion

By comparing the two-dimensional gel electrophoresis (2-DE) spot patterns from different samples, changes in individual proteins can be detected and quantified. This also allows identification of proteins that are characteristic of a specific subcellular fraction. In the present study, subcellular adipocyte fractionation in different animals resulted in a similar protein pattern of each fraction. In addition, different band fingerprinting of each fraction obtained by 2-DE procedure assured that each fraction contained different protein patterns, thus demonstrating that the enzyme assays were not cross-contaminated due to mixture of fractions. The effectiveness of this subcellular adipocyte fractionation was previously demonstrated only by the use of markers as cytochrome C reductase for HDM, galactosyltransferase for LDM, and 5’-nucleotidase for MF (Simpson et al. 1983, Maianu et al. 2001).

IRAP is a member of the M1 family of zinc-dependent metalloproteinases (Maianu et al. 2001, Ye et al. 2008) that has been widely studied in CNS and adipocytes (Keller et al. 1995, Keller 2004, Albiston et al. 2010, Jordens et al. 2010, Bogan 2012). In adipocytes, the regulation of its traffic from LDM toward MF by insulin (Keller et al. 1995, Keller 2004, Jordens et al. 2010) allows catalytic or receptor roles of this protein in extracellular environment. Most of the literature reports in this sense assess this traffic by immunoblotting or immunofluorescence (Jordens et al. 2010, Hirata et al. 2011, Bogan 2012) without concomitant evaluation of catalytic activity. When catalytic activity is measured, aminoacyl substrates with N-terminal Leu are usually used (Lew et al. 2003, Saníger et al. 2011). The present study showed only a trend, without statistical significance, for insulin-stimulated traffic of LeuAP activity in adipocytes, which did not change as a function of metabolic challenges applied. However, insulin effectively stimulates CysAP activity trafficking from LDM toward MF in all animal groups under study. In this way, it fits in the current functional concept of IRAP. It was also noteworthy that the metabolic challenge posed by MSG obesity interferes directly on CysAP activity, influencing its anterograde traffic, independently of insulin stimulation. The insulin-dependent traffic of CysAP activity has important pathophysiological implications, since endogenous substrates with N-terminal cysteine pair susceptible to hydrolysis by this enzyme activity, that are available in the extracellular environment, have consistent and comprehensive roles related to energy metabolism. One substrate is AVP, which plays an antilipolytic role in adipocyte (Hiroyama et al. 2007), besides antidiuretic, vasoconstrictor (Küchler et al. 2010) and a memory facilitator agent (Ramanathan et al. 2012). Another is OXT, best known for its role in lactation and parturition (Caldwell et al. 1990), but also known as an anorexigenic agent, reducing meal size and intake of highly palatable foods, such as sucrose (Maejima et al. 2011) and increasing latency to the first meal (Blevins & Ho 2013). In addition, OXT has been recognized as a facilitator of interpersonal attraction (Scheele et al. 2012, 2013), providing relaxed physiological states (Carter & Porges 2013). Thus, insulin-independent stimulation of CysAP activity traffic from LDM toward MF is a deleterious dysregulation in MSG obesity, since it facilitates extracellular hydrolytic action of IRAP normally under insulin control on peptides whose actions contribute to homeostasis.

In conclusion, catalytic preference and insulin-regulated traffic of IRAP in adipocytes that is disturbed by obesity are interrelated with comprehensive roles of endogenous substrates with N-terminal cysteine pair in energy metabolism homeostasis.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding

This work was supported by a Research Grant number 2010/01385-5 from FAPESP (Sao Paulo Research Foundation, Brazil). P F S was the recipient of a CNPq (National Council for Scientific and Technological Development, Brazil) productivity grant number (PQ-302533/2011-7). R F A was the recipient of a CAPES (Coordination for the Improvement of Higher Education Personnel of the Ministry of Education in Brazil) fellowship (DR-2008-2012). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Author contribution statement

P F S conceived and R F A, L G V and N Y carried out experiments, P F S and R F A analyzed data. P F S and R F A were involved in writing the paper. All authors had final approval of the submitted and published versions. All authors have read and agree with the manuscript as written.

Acknowledgements

The authors thank all the staff at the Unit of Translational Endocrine Pharmacology and Physiology at Laboratory of Pharmacology for technical support, and the agency GLMS Scientific Language Services for having improved the English in this manuscript.

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  • KellerSRScottHMMastickCCAebersoldRLienhardGE1995Cloning and characterization of a novel insulin-regulated membrane aminopeptidase from Glut4 vesicles. Journal of Biological Chemistry2702361223618. (doi:10.1074/jbc.270.40.23612).

    • Search Google Scholar
    • Export Citation
  • KüchlerSPerwitzNSchickRRKleinJWestphalS2010Arginine-vasopressin directly promotes a thermogenic and pro-inflammatory adipokine expression profile in brown adipocytes. Regulatory Peptides164126132. (doi:10.1016/j.regpep.2010.05.016).

    • Search Google Scholar
    • Export Citation
  • LewRAMustafaTYeSMcDowallSGChaiSYAlbistonAL2003Angiotensin AT4 ligands are potent, competitive inhibitors of insulin regulated aminopeptidase (IRAP). Journal of Neurochemistry86344350. (doi:10.1046/j.1471-4159.2003.01852.x).

    • Search Google Scholar
    • Export Citation
  • LoKALabadorfAKennedyNJHanMSYapYSMatthewsBXinXSunLDavisRJLodishHF2013Analysis of in vitro insulin-resistance models and their physiological relevance to in vivo diet-induced adipose insulin resistance. Cell Reports5259270. (doi:10.1016/j.celrep.2013.08.039).

    • Search Google Scholar
    • Export Citation
  • LunaMSValenteRHPeralesJVieiraMLYamanouyeN2013Activation of Bothrops jararaca snake venom gland and venom production: a proteomic approach. Journal of Proteomics94460472. (doi:10.1016/j.jprot.2013.10.026).

    • Search Google Scholar
    • Export Citation
  • MaejimaYIwasakiYYamaharaYKodairaMSedbazarUYadaT2011Peripheral oxytocin treatment ameliorates obesity by reducing food intake and visceral fat mass. Aging311691177.

    • Search Google Scholar
    • Export Citation
  • MaianuLKellerSRGarveyWT2001Adipocytes exhibit abnormal subcellular distribution and translocation of vesicles containing glucose transporter 4 and insulin-regulated aminopeptidase in type 2 diabetes mellitus: implications regarding defects in vesicle trafficking. Journal of Clinical Endocrinology and Metabolism8654505456. (doi:10.1210/jcem.86.11.8053).

    • Search Google Scholar
    • Export Citation
  • McKeelDWJarettL1970Preparation and characterization of a plasma membrane fraction from isolated fat cells. Journal of Cell Biology44417432. (doi:10.1083/jcb.44.2.417).

    • Search Google Scholar
    • Export Citation
  • NakagawaTUkaiKOhyamaTGomitaYOkamuraH2000Effects of chronic administration of sibutramine on body weight, food intake and motor activity in neonatally monosodium glutamate-treated obese female rats: relationship of antiobesity effect with monoamines. Experimental Animals49239249. (doi:10.1538/expanim.49.239).

    • Search Google Scholar
    • Export Citation
  • RamanathanGCilzNIKuradaLHuBWangXLeiS2012Vasopressin facilitates GABAergic transmission in rat hippocampus via activation of V(1A) receptors. Neuropharmacology6312181226. (doi:10.1016/j.neuropharm.2012.07.043).

    • Search Google Scholar
    • Export Citation
  • RodbellM1964Metabolism of isolated fat cells. Journal of Biological Chemistry239375380.

  • RossSAScottHMMorrisNJLeungWYMaoFLienhardGEKellerSR1996Characterization of the insulin-regulated membrane aminopeptidase in 3T3-L1 adipocytes. Journal of Biological Chemistry27133283332. (doi:10.1074/jbc.271.6.3328).

    • Search Google Scholar
    • Export Citation
  • RusterCWolfG2013The role of the renin-angiotensin-aldosterone system in obesity-related renal diseases. Seminars in Nephrology334453. (doi:10.1016/j.semnephrol.2012.12.002).

    • Search Google Scholar
    • Export Citation
  • SanígerMARamirez-ExpositoMJDe La ChicaSCarrera-GonzálezMPMayasMDMartínez-MartosJM2011α-1-adrenergic receptor blockade modifies insulin-regulated aminopeptidase (IRAP) activity in rat prostate and modulates oxytocin functions. Drug Metabolism Letters5192196. (doi:10.2174/187231211796905035).

    • Search Google Scholar
    • Export Citation
  • SanoMItakuraAItoMTakeuchiMOkadaMKotaniTMizutaniSKikkawaF2005Placental leucine aminopeptidase might regulate the effects of oxytocin with resolution in endothelial cells. Medical Science Monitor11BR195BR199.

    • Search Google Scholar
    • Export Citation
  • ScheeleDStriepensNGüntürkünODeutschländerSMaierWKendrickKMHurlemannR2012Oxytocin modulates social distance between males and females. Journal of Neuroscience321607416079. (doi:10.1523/JNEUROSCI.2755-12.2012).

    • Search Google Scholar
    • Export Citation
  • ScheeleDWilleAKendrickKMStoffel-WagnerBBeckerBGüntürkünOMaierWHurlemannR2013Oxytocin enhances brain reward system responses in men viewing the face of their female partner. PNAS1102030820313. (doi:10.1073/pnas.1314190110).

    • Search Google Scholar
    • Export Citation
  • SimpsonIAYverDRHissinPJWardzalaLJKarnieliESalansLBCushmanSW1983Insulin-stimulated translocation of glucose transporters in the isolated rat adipose cells: characterization of subcellular fractions. Biochimica et Biophysica Acta763393407. (doi:10.1016/0167-4889(83)90101-5).

    • Search Google Scholar
    • Export Citation
  • TesauroMCardilloC2011Obesity, blood vessels and metabolic syndrome. Acta Physiologica203279286. (doi:10.1111/j.1748-1716.2011.02290.x).

    • Search Google Scholar
    • Export Citation
  • WatsonRTPessinJE2001Subcellular compartmentalization and trafficking of the insulin-responsive glucose transporter, GLUT4. Experimental Cell Research2717583. (doi:10.1006/excr.2001.5375).

    • Search Google Scholar
    • Export Citation
  • YeSChaiSYLewRAAscherDBMortonCJParkerMWAlbistonAL2008Identification of modulating residues defining the catalytic cleft of insulin-regulated aminopeptidase. Biochemistry and Cell Biology86251261. (doi:10.1139/O08-037).

    • Search Google Scholar
    • Export Citation
  • ZeydaMStulnigTM2009Obesity, inflammation, and insulin resistance – a mini-review. Gerontology55379386. (doi:10.1159/000212758).

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    Representative 2-DE gel images of (i) high (HDM) and (ii) low (LDM) density microsomal and (iii) plasma membrane (MF) fractions from isolated adipocytes of three healthy animals. Samples (80 μg) were applied to 3–10 IPG strips followed by electrophoresis on 12.5% acrylamide gels. Spots are designated A to L.

  • View in gallery

    Leucyl-aminopeptidase (LeuAP) activity of high (HDM) and low (LDM) density microsomal and plasma membrane (MF) fractions from isolated adipocytes of retroperitoneal fat pad of control (C), food deprived (FD) and obese (MSG) rats in basal condition or under insulin stimulation. Values are mean±s.e.m. from triplicates of n animals (n in parenthesis over the bars). ANOVA, HDM vs LDM vs MF: basal (C, P=0.5125; FD, P=0.7072; MSG, P=0.6882); insulin (C, P=0.8962; FD, P=0.2894; MSG, P=0.2511).

  • View in gallery

    Cystyl-aminopeptidase (CysAP) activity of high (HDM) and low (LDM) density microsomal and plasma membrane (MF) fractions from isolated adipocytes of retroperitoneal fat pad of control (C), food deprived (FD) and obese (MSG) rats in basal condition or under insulin stimulation. Values are mean±s.e.m. from triplicates of n animals (n in parenthesis over the bars). ANOVA, HDM vs LDM vs MF: basal (C, P=0.3973; FD, P=0.8675; MSG, P=0.0095); insulin (C, P=0.0031; FD, P=0.0049; MSG, P=0.0118. Multiple comparison Tukey test (P<0.05, different letters in the same group and stimulus indicate significant differences).

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    • Search Google Scholar
    • Export Citation
  • KüchlerSPerwitzNSchickRRKleinJWestphalS2010Arginine-vasopressin directly promotes a thermogenic and pro-inflammatory adipokine expression profile in brown adipocytes. Regulatory Peptides164126132. (doi:10.1016/j.regpep.2010.05.016).

    • Search Google Scholar
    • Export Citation
  • LewRAMustafaTYeSMcDowallSGChaiSYAlbistonAL2003Angiotensin AT4 ligands are potent, competitive inhibitors of insulin regulated aminopeptidase (IRAP). Journal of Neurochemistry86344350. (doi:10.1046/j.1471-4159.2003.01852.x).

    • Search Google Scholar
    • Export Citation
  • LoKALabadorfAKennedyNJHanMSYapYSMatthewsBXinXSunLDavisRJLodishHF2013Analysis of in vitro insulin-resistance models and their physiological relevance to in vivo diet-induced adipose insulin resistance. Cell Reports5259270. (doi:10.1016/j.celrep.2013.08.039).

    • Search Google Scholar
    • Export Citation
  • LunaMSValenteRHPeralesJVieiraMLYamanouyeN2013Activation of Bothrops jararaca snake venom gland and venom production: a proteomic approach. Journal of Proteomics94460472. (doi:10.1016/j.jprot.2013.10.026).

    • Search Google Scholar
    • Export Citation
  • MaejimaYIwasakiYYamaharaYKodairaMSedbazarUYadaT2011Peripheral oxytocin treatment ameliorates obesity by reducing food intake and visceral fat mass. Aging311691177.

    • Search Google Scholar
    • Export Citation
  • MaianuLKellerSRGarveyWT2001Adipocytes exhibit abnormal subcellular distribution and translocation of vesicles containing glucose transporter 4 and insulin-regulated aminopeptidase in type 2 diabetes mellitus: implications regarding defects in vesicle trafficking. Journal of Clinical Endocrinology and Metabolism8654505456. (doi:10.1210/jcem.86.11.8053).

    • Search Google Scholar
    • Export Citation
  • McKeelDWJarettL1970Preparation and characterization of a plasma membrane fraction from isolated fat cells. Journal of Cell Biology44417432. (doi:10.1083/jcb.44.2.417).

    • Search Google Scholar
    • Export Citation
  • NakagawaTUkaiKOhyamaTGomitaYOkamuraH2000Effects of chronic administration of sibutramine on body weight, food intake and motor activity in neonatally monosodium glutamate-treated obese female rats: relationship of antiobesity effect with monoamines. Experimental Animals49239249. (doi:10.1538/expanim.49.239).

    • Search Google Scholar
    • Export Citation
  • RamanathanGCilzNIKuradaLHuBWangXLeiS2012Vasopressin facilitates GABAergic transmission in rat hippocampus via activation of V(1A) receptors. Neuropharmacology6312181226. (doi:10.1016/j.neuropharm.2012.07.043).

    • Search Google Scholar
    • Export Citation
  • RodbellM1964Metabolism of isolated fat cells. Journal of Biological Chemistry239375380.

  • RossSAScottHMMorrisNJLeungWYMaoFLienhardGEKellerSR1996Characterization of the insulin-regulated membrane aminopeptidase in 3T3-L1 adipocytes. Journal of Biological Chemistry27133283332. (doi:10.1074/jbc.271.6.3328).

    • Search Google Scholar
    • Export Citation
  • RusterCWolfG2013The role of the renin-angiotensin-aldosterone system in obesity-related renal diseases. Seminars in Nephrology334453. (doi:10.1016/j.semnephrol.2012.12.002).

    • Search Google Scholar
    • Export Citation
  • SanígerMARamirez-ExpositoMJDe La ChicaSCarrera-GonzálezMPMayasMDMartínez-MartosJM2011α-1-adrenergic receptor blockade modifies insulin-regulated aminopeptidase (IRAP) activity in rat prostate and modulates oxytocin functions. Drug Metabolism Letters5192196. (doi:10.2174/187231211796905035).

    • Search Google Scholar
    • Export Citation
  • SanoMItakuraAItoMTakeuchiMOkadaMKotaniTMizutaniSKikkawaF2005Placental leucine aminopeptidase might regulate the effects of oxytocin with resolution in endothelial cells. Medical Science Monitor11BR195BR199.

    • Search Google Scholar
    • Export Citation
  • ScheeleDStriepensNGüntürkünODeutschländerSMaierWKendrickKMHurlemannR2012Oxytocin modulates social distance between males and females. Journal of Neuroscience321607416079. (doi:10.1523/JNEUROSCI.2755-12.2012).

    • Search Google Scholar
    • Export Citation
  • ScheeleDWilleAKendrickKMStoffel-WagnerBBeckerBGüntürkünOMaierWHurlemannR2013Oxytocin enhances brain reward system responses in men viewing the face of their female partner. PNAS1102030820313. (doi:10.1073/pnas.1314190110).

    • Search Google Scholar
    • Export Citation
  • SimpsonIAYverDRHissinPJWardzalaLJKarnieliESalansLBCushmanSW1983Insulin-stimulated translocation of glucose transporters in the isolated rat adipose cells: characterization of subcellular fractions. Biochimica et Biophysica Acta763393407. (doi:10.1016/0167-4889(83)90101-5).

    • Search Google Scholar
    • Export Citation
  • TesauroMCardilloC2011Obesity, blood vessels and metabolic syndrome. Acta Physiologica203279286. (doi:10.1111/j.1748-1716.2011.02290.x).

    • Search Google Scholar
    • Export Citation
  • WatsonRTPessinJE2001Subcellular compartmentalization and trafficking of the insulin-responsive glucose transporter, GLUT4. Experimental Cell Research2717583. (doi:10.1006/excr.2001.5375).

    • Search Google Scholar
    • Export Citation
  • YeSChaiSYLewRAAscherDBMortonCJParkerMWAlbistonAL2008Identification of modulating residues defining the catalytic cleft of insulin-regulated aminopeptidase. Biochemistry and Cell Biology86251261. (doi:10.1139/O08-037).

    • Search Google Scholar
    • Export Citation
  • ZeydaMStulnigTM2009Obesity, inflammation, and insulin resistance – a mini-review. Gerontology55379386. (doi:10.1159/000212758).

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