Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

Obesity is associated with profound alterations of cardiovascular functions including an increase in blood pressure. Elevated plasma leptin levels, a hormone mainly produced by the adipose tissue, is occurs frequently in obesity and related disorders in humans (Caro et al., 1996). Initially considered as a satiety factor, leptin also exerts sympathetic, renal, and metabolic effects that could contribute to the altered cardiovascular functions occurring in obesity (Haynes et al.,1997, Mark et al., 1999). Leptin can affect blood pressure through two opposite mechanisms: 1) enhancement of the sympathetic tone (Haynes et al.,1997), leading to an increase in blood pressure; and 2) promotion of natriuresis (Jackson et al., 1997) and nitric oxide vasorelaxant effect (Lembo et al., 2000; Vecchione et al.,2002), which both contribute to lower blood pressure. However, chronic effect of leptin appears to be predominately pressor. Indeed infusion of leptin at rates that raise plasma leptin to levels similar to those found in obesity, increases mean arterial pressure (Shek et al., 1998). Furthermore transgenic mice overexpressing leptin develop elevation of blood pressure (Aizawa Abe et al., 2000). Elevated plasma leptin concentrations have also been described in hypertensive patients (Agata et al., 1997). Leptin could therefore be one of the pathophysiological candidates linking obesity to hypertension (Mark et al., 1999; Aizawa Abe et al., 2000; Hall et al., 2000; Ogawa et al., 2002).

Leptin acts through binding to leptin receptor, Ob-R, which is expressed as various splice variants and is a member of the extended class I cytokine receptor family. Both the short Ob-Ra isoform and the full-length Ob-Rb variant are expressed in the kidney (Hoggard et al., 1997). Acute administration of leptin acts on the kidney to promote natriuresis and diuresis (Jackson et al., 1997; Serradeil-Le Gal et al., 1997). In the obese Zucker rat, an attenuated diuretic and natriuretic response to systemic leptin infusion has been described (Villareal et al., 1998). Because obese Zucker rats have a mutation in the Ob-R (Takaya et al., 1996) leading to reduced signal transduction, renal leptin resistance may explain the attenuated natriuretic and diuretic response observed in this strain. Localization studies showing existence of the Ob-R in the renal inner medulla (Hoggard et al., 1997; Serradeil-Le Gal et al., 1997) further support the requirement of the receptor in the mechanisms whereby leptin stimulates natriuresis. It must be noted that the elevated blood pressure in obese Zucker rats compared with lean control rats was associated with a marked increase in sodium reabsorption (Alonso-Galicia et al., 1996). As obesity is associated with resistance to leptin effects, the occurrence of leptin resistance in the kidney can also be reasonably postulated in the obese state and thus can contribute at least partly to the obesity hypertension. We have previously shown that overweight induced by cafeteria diet feeding is associated with sexual dimorphism in the development of hypertension (Coatmellec-Taglioni et al., 2000, 2002). Indeed, male cafeteria-fed rats developed hypertension, whereas female rats fed the same diet are overweight but normotensive (Coatmellec-Taglioni et al., 2002). Moreover this sexual dimorphism of the cafeteria diet-induced hypertension was reversed by testosterone imprinting of female rats at birth (Plut et al., 2002). The aim of the present study was to investigate the renal leptin receptor in hypertension induced by cafeteria feeding. Therefore, renal leptin receptor mRNA and binding capacities were analyzed in male and intact or neonatally androgenized female cafeteria-fed rats. We show here that renal Ob-R down-regulation only appears in hypertensive rats.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Animal Procedures. Testosterone treatment: female Sprague-Dawley rat pups were injected subcutaneously with 1 mg of testosterone propionate in olive oil at birth (Plut et al., 2002). Cafeteria diet: male, female and testosterone-treated female Sprague-Dawley rats with an average weight of 100 g were divided into two groups and maintained at room temperature with a 12-h light/dark cycle. The first group, "control" received standard chow (Usine d'Alimentation Rationnelle, Villemoisson sur Orge, France; 25% protein, 6% fat, 69% carbohydrate) ad libitum. The second group, "cafeteria", was given four palatable foods for human consumption in addition to the chow (Mandenoff et al., 1982). The four supplementary items were changed every day on a weekly rotation. The lists of palatable items included ham, salami, potato chips, marshmallows, cheese, cookies, bread, noodles, and a large choice of candies and chocolate bars. The average composition of the diet was 15% protein, 48% fat and 36% carbohydrate (Mandenoff et al., 1982).

Blood Pressure Measurement. Systolic blood pressure was measured between 10:00 and 12:00 AM using the tail-cuff method, with an electrosphygmomanometer (Physiograph MK III; Narco-Bio-Systems Inc., Houston, TX), on unanesthesized, restrained rats warmed to 38°C for 10 min. At least, five replicate blood pressure measurements were obtained on 2 consecutive days. The average of all measurements for each rat was taken as representative of systolic blood pressure. Two days later, fasted rats were killed by decapitation, and their kidneys carefully removed. Renal medulla was rapidly dissected and frozen in liquid nitrogen. All experimental protocols were approved by the University Animal Use and Care Committee.

Hormonal Determinations. Plasma leptin and insulin levels were measured by radioimmunoassay using commercial kits provided by LINCO Research, Inc. (St. Charles, MO) with rat leptin and insulin as standards.

Membrane Preparation. Renal medulla was homogenized in a medium containing 250 mM sucrose, 5 mM Tris-HCl, pH 8.0, 3 mM MgCl₂, and 1 mM EDTA. The 300 g pellet was collected and suspended in a buffer made hypotonic by dilution of sucrose. This washing procedure was repeated three times, and the final pellet was suspended in the same medium. Protein concentration was determined by the method of Bradford using bovine serum albumin as a standard.

Binding Assays. Medullary membranes (150 µg) were incubated for 4 h at 4°C with ¹²⁵I-leptin (81.4 × 10¹² Bq/mmol; PerkinElmer Life Sciences, Boston, MA), 141 mM NaCl, 15 mM Hepes/NaOH, 4 mM KCl, 1.4 mM CaCl₂, 5% bovine serum albumin, and 300 µg/ml bacitracin in a final volume of 200 µl. Reactions were stopped by dilution with ice-cold incubation buffer and rapid vacuum filtration through Whatman GF/C filters previously soaked in 0.5% polyethylenimine. The filters were washed twice with 4 ml of ice-cold incubation buffer. The radioactivity retained on filters was quantified by gamma counting. Nonspecific binding was determined in the presence of 0.3 mM unlabeled leptin (PeproTech Inc., Rocky Hill, NJ). For competition studies, membranes were incubated with 0.12 nM ¹²⁵I-leptin and 0.003 to 300 nM unlabeled leptin for 4 h at 4°C. Data for equilibrium dissociation constant (K_d), maximum binding capacities (B_max), concentrations of unlabeled leptin required to obtain 50% inhibition of the specific binding (IC₅₀), and inhibition constant (K_i) were determined using a nonlinear least squares curve-fitting program (GraphPad PRISM; GraphPad Software Inc., San Diego, CA).

Western Blot Analysis. Membrane preparations were diluted in an equal volume of 2× Laemmli's SDS-polyacrylamide gel electrophoresis sample buffer (20% glycerol, 4% SDS, 10% 2-mercaptoethanol, 100 mM Tris-HCl, and 0.02% bromphenol blue). Samples were boiled at 95°C for 5 min, subjected to 7.5% SDS-polyacrylamide gel electrophoresis, and transferred to nitrocellulose filters. Ob-R was detected with a goat anti-Ob-R polyclonal antiserum (K-20) (Santa Cruz Biotechnology Inc., Santa Cruz, CA) and horseradish peroxidasse-conjugated rabbit anti-goat IgG antibody. Immunoreactions were detected with enhanced chemiluminescence (ECL; Amersham Pharmacia Biotech, Saint Quentin en Yveline, France).

Expression of Leptin Receptor mRNA. Total RNA from renal medulla was isolated using TRIzol reagent, and cDNA was generated using Superscript II reverse transcriptase protocol (Invitrogen, Carlsbad, CA). Briefly, mRNA was isolated by incubating 1.5 µg of total RNA for 10 min at 70°C in the presence of 0.5 µg of oligo(dT)_12-18. cDNA synthesis was conducted for 50 min at 42°C with 0.1 mol/l dithiothreitol, 10 mM dNTP, and 200 units of Superscript II followed by heat inactivation at 70°C for 15 min. Reverse transcription products (10%) were amplified in PCR mixture containing 200 µM of each dNTP, 0.5 mM of each primer, 1 µCi [³H]dCTP (1.96 × 10¹² Bq/mmol; Amersham, Little Chalfont, England), and 2.5 units HotStarTaq DNA polymerase (QIAGEN, Courtaboeuf, France). After initial activation of the HotStarTaq DNA polymerase for 15 min at 95°C, 26 amplification cycles were performed for 1 min at 94°C, 1 min at 53°C, and 1 min at 72°C for -actin, 35 cycles performed for Ob-Ra and 40 cycles for 1 min at 94°C, 1 min at 55°C, and 1 min at 72°C for Ob-Rb. Primers used for amplification (Eurogentec, Herstal, Belgium) were described elsewhere [-actin (Plut et al., 2002), Ob-Ra (Takaya et al., 1996), Ob-Rb (Bjorbaek et al., 1998)]. Linear ranges for each leptin receptor isoform and actin were determined to ensure that all reactions were analyzed during the exponential phase of amplification in all groups. PCR products were separated by agarose gel electrophoresis, bands of interest were cut off the gel, solubilized, and quantified by liquid scintillation counting. Reported data were corrected for -actin values and expressed as percentage of control values.

Statistical analysis. All results were expressed as the mean ± S.E.M. For binding studies, statistical analysis was performed using analysis of variance. Other data were analyzed using Student's t test (p < 0.05 was considered statistically significant).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Animal Characteristics. As shown in Table 1, after 10 weeks of cafeteria diet feeding, fat pad weight was increased in male, untreated female, and testosterone-treated female rats. As previously observed, male cafeteria-fed rats were hypertensive, whereas female cafeteria-fed rats remained normotensive (Coatmellec-Taglioni et al., 2000, 2001). However, when female rats treated with testosterone at birth are fed with a cafeteria diet, they share an increase in blood pressure similar to male rats. Fasting plasma insulin levels were higher in the cafeteria-fed animals than in the group on regular chow. Moreover, an increase in plasma leptin level was detected in cafeteria groups.

Binding Studies. Whereas binding leptin can be detected in cultured glomerular endothelial cells (Wolf et al., 1999), specific ¹²⁵I-leptin binding was almost undetectable in crude cortical preparation from rat kidney as observed by Serradeil Le Gal et al. (1997). Therefore, we investigate Ob-R binding sites in crude membranes from the renal medulla. The expression of Ob-R protein in these crude membrane preparations was demonstrated by Western blot analyses using an anti-Ob-R goat polyclonal antibody (Fig. 1). The major protein detected had a molecular mass of ~120 kDa, and this immunoreactive band completely disappeared in the presence of the specific peptide (Fig. 1). Using the same antibody, Shioda et al. (1998) detected a major 120-kDa band in the rat hypothalamus where Ob-Ra and ObRb are expressed. Thus, our result does not exclude the presence of Ob-Rb in membranes of renal medulla.

View larger version (36K): [in this window] [in a new window]   Fig. 1.   Leptin receptors in renal medulla from male (lanes 1 and 3) and female (lanes 2 and 4) rats. Membrane samples prepared for binding experiments were analyzed by Western blot analysis using an anti-Ob-R polyclonal antibody. A protein with a molecular mass of ~ 120 kDa was detected (lanes 1 and 2) and was fully blunted by the use of a specific peptide (lanes 3 and 4). These data are representative of five experiments.

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Effects of cafeteria diet on leptin binding to medullary membranes from rat kidneys. Renal medullary membranes from male (A), female (B), or testosterone-treated female (C), control () or cafeteria () rats were incubated in 125I-leptin in the absence or presence of unlabeled leptin. Data shown are from one experiment representative of five separate experiments.

View larger version (13K): [in this window] [in a new window]   Fig. 3.   Inhibition of 125I-leptin-specific binding to renal medullary membranes from male (A), female (B), or testosterone-treated female (C) cafeteria-fed rats. Membranes from control () or cafeteria () rats were incubated with 125I-leptin in the presence of increasing concentrations of unlabeled leptin (from 0.03 to 300 nM). Data shown are from one experiment representative of five separate experiments.

Expression of Long and Short Leptin Receptor mRNA. To determine whether the effect of cafeteria feeding on ¹²⁵I-leptin binding capacities was associated with parallel changes in Ob-R expression levels, reverse transcriptase-PCR analysis of Ob-Ra and Ob-Rb was performed in all experimental groups. As shown in Fig. 4, A and B, no differences occurred in the amount of either Ob-Ra and Ob-Rb mRNA between the control and the cafeteria-fed groups, regardless of sex. Cafeteria diet did not alter Ob-Ra and Ob-Rb mRNA expression in the kidney of testosterone-treated female rats either (90 ± 12 and 85 ± 17 of respective control mRNA level). Moreover, no differences in the size of PCR products were observed between control and cafeteria-fed rats, ruling out the presence of alternative splicing in cafeteria-fed animals, as observed in some genetic forms of obesity (Fig. 4A). Finally, the fragment generated from -actin primers, which was chosen to span 2 introns, was the only amplified fragment suggesting no genomic DNA contamination (Fig. 4A). Thus, the Ob-R down-regulation found in the kidney of male and testosterone-treated female rats is not related to a decrease in Ob-R mRNA expressions.

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Effect of cafeteria diet feeding on the relative Ob-Ra and Ob-Rb mRNA levels in the renal medulla of male or female Sprague-Dawley rats. A, representative gel electrophoresis of reverse transcriptase-PCR products, using -actin-, Ob-Ra-, or Ob-Rb-specific primers in kidney medulla from control (1 and 3) and cafeteria (2 and 4) male and female rats. Representative autoradiographies shown are from one experiment representative of five. B, expression of Ob-Ra and Ob-Rb mRNA from kidney medulla of control () or cafeteria () rats. Values are expressed as the percentage of the control mRNA level determined by liquid scintillation counting and normalized to -actin.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The present study demonstrates that the sexual dimorphism in cafeteria diet-induced hypertension is associated with the same dimorphism in Ob-R down-regulation. Furthermore, neonatal testosterone treatment allowed the development of hypertension in female rats fed with the cafeteria diet. These hypertensive female rats also elicit renal Ob-R down-regulation as do hypertensive male rats.

Although the mechanisms responsible for the gender differences in hypertension are not clear, there is significant evidence that androgens such as testosterone play an important role in the gender-specific blood pressure regulation (Reckelhoff, 2001). Indeed, hypertension was reduced by castration in SHR and in Dahl and Sabra salt-sensitive rats (Crofton et al., 1993; Gong et al., 1994; Khalid et al., 2001). Furthermore, administration of androgen receptor antagonist, for 10 days after birth, attenuates the hypertension of male adult SHR, whereas neonatal androgenization of female SHR leads to similar hypertension as that in male (Cambotti et al., 1984). All together, these results strengthen the influence of testosterone on the promotion of hypertension. Neonatal testosterone induces specific brain maturation, which may promote sensitivity to cafeteria diet, leading to the onset of hypertension.

Leptin receptor mRNA and/or protein down-regulation has been clearly characterized in mouse brain (Lin et al., 2000), rat hypothalami (Martin et al., 2000) after either high-fat diet or leptin loads. In these previous studies, Ob-R down-regulation was attributed to altered transcription of the receptor gene or mRNA stability. In contrast, the present study demonstrates an obvious leptin receptor protein down-regulation in renal medullary membranes from male cafeteria-fed rats, without changes in Ob-Ra and Ob-Rb mRNA levels. One common well characterized mechanism by which cell surface receptor number can decrease is the ligand-induced receptor internalization and degradation by lysosomal pathway. Two independent groups have recently demonstrated that leptin is able to down-regulate its own receptor through a coat-pitted-dependent mechanism (Barr et al., 1999; Uotani et al., 1999). Because the presently reported decrease in available cell surface Ob-R is inversely correlated to plasma leptin level in the kidney of male rats and testosterone-treated female rats, the receptor down-regulation could be related to excess leptin levels. However, despite a similar rise in plasma leptin levels, renal Ob-R were not down-regulated in female rat kidneys. This suggested the existence of gender-specific leptin sensitivity. The kidney has been shown to be the main site of leptin clearance, where leptin is degraded (Cumin et al., 1997) by a receptor-mediated internalization through a clathrin-mediated mechanism (Barr et al., 1999; Uotani et al., 1999). Because males had greater renal leptin clearance than females (Meyer et al., 1997), more leptin may access to renal Ob-R in male and induce the greatest Ob-R down-regulation. Furthermore androgens, in vivo as in vitro, increase clathrin expression (Prescott et Tindall, 1998) and may therefore participate in the greatest sensitivity of male to Ob-R down-regulation. Gender is also a major determinant of plasma leptin in human (Considine et al., 1996). Although women are likely to have a greater body fat mass than men, leptin levels rose more rapidly as a function of body mass index in women than in men (Kennedy et al., 1997). Therefore, higher levels of leptin are required in women to achieve similar biological endpoints. This suggests that there may be a difference in the biological homeostatic set point for obesity in women. Soluble leptin receptor is the major leptin-binding protein in the plasma (Lammert et al., 2001). Obesity is associated with decreasing levels of circulating soluble leptin receptor, but a gender difference was observed in soluble leptin receptor levels, which were higher in obese men than in obese women (Ogier et al., 2002). Overexpression of soluble leptin receptor leads to an improved weight-reducing effect of leptin in ob/ob mice (Huang et al., 2001) suggesting that these receptors affect bioavailability and functionality of leptin. Therefore, an eventual gender difference in soluble leptin receptors in cafeteria-fed rats can be responsible for renal leptin sensitivity.

On the other hand, because leptin receptor down-regulation only occurred in hypertensive rats, these alterations might have also appeared as a consequence of renal disease induced by hypertension. Indeed, the kidney is one of the main sites of blood pressure control, and hypertension is generally associated with renal damage (Guyton et al., 1981). Moreover, male animals are at greatest risk of developing renal diseases and exhibit a more rapid progression of renal injury (Reckelhoff and Granger, 1999). Whatever the causes of the renal Ob-R down-regulation, this alteration may reduce natriuresis and renal nitric oxide production, which both contribute to leptin hypotensive actions (Jackson and Li, 1997; Vecchione et al., 2002). In summary, this study provides some evidence that renal leptin receptor down-regulation is associated with a diet-induced hypertension in Sprague-Dawley rats. This effect does not appear in the kidney of female cafeteria-fed rats. Thus, sexual dimorphism in renal leptin receptor sensitivity and hypertension occurs in Sprague-Dawley rats.