Oleoyl-estrone metabolic effects in relation with caloric restriction in inbred Beta rats with spontaneous obesity and type 2 diabetes.

Marta  D. Posadas¹, María C. Olguín², María I. Zingale², Gilda Revelant², Verónica Labourdette¹, María del C. Gayol¹, Susana Calderari¹

¹ Cátedra de Biología, Facultad de Ciencias Médicas,
² Cátedra de Bromatología, Facultad de Ciencias Bioquímicas y Farmacéuticas,  Universidad Nacional de Rosario

Postal address: Dra. María Catalina Olguin, Cátedra de Bromatología, Facultad de Ciencias Bioquímicas y Farmacéuticas, Suipacha 531, 2000 Rosario, Argentina. Fax: (54-0341). E-mail: molguin@fbioyf.unr.edu.ar

Abstract
Spontaneously hypertriacylglycerolemic obese and diabetic inbred IIM Beta rats were treated with oleoyl- estrone for 10 days. Pair-feeding was performed to determine some oleoyl-estrone effects dependent on the caloric restriction it promotes. Twenty-five 200 day-old Beta males receiving a daily gavage of 0.2 ml sunflower oil were divided into the following groups: 1) daily dose of 10 nmol/g oleoyl-estrone; 2) pair-fed; 3) control. The variables measured were: whole body protein, water and lipid; retroperitoneal and epididymal fat depot weights; plasma urea, glucose, insulin, triacylglycerols and cholesterol. Biomass and food intake were assessed daily. Oleoyl-estrone and pair-fed groups expressed similar variations in body composition and significant body weight losses due to reduction in food intake. Oleoyl-estrone and pair-fed treatments significantly reduced retroperitoneal fat depot weights, but not epididymal ones. In oleoyl-estrone and pair-fed groups hyperglycemia decreased and insulinemia lowered significantly. Plasma normal total cholesterolemia and hypertriacylglycerolemia values typical of Beta rats decreased strongly compared to controls, though attaining significantly different values between oleoyl-estrone and pair-fed groups. Plasma total cholesterol appeared as more  sensitive to caloric restriction than triacylglycerols through a specific oleoyl-estrone- mediated effect.

Key words: Oleoyl-estrone; Inbred Beta rats; Obesity; Slimming; Diabetes.

Resumen
Efectos metabólicos de la oleoil-estrona en relación con la restricción calórica en ratas Beta endocriadas, con obesidad espontánea y diabetes tipo II. Ratas endocriadas de la línea IIMBeta con obesidad, hipertriacilglicerolemia y diabetes espontáneas fueron tratadas con oleoil-estrona durante 10 días. Un grupo con restricción alimentaria fue incluido en el estudio a fin de aislar algunos efectos de la oleoil-estrona dependientes de la restricción calórica que ésta promueve. Veinticinco ratas Beta macho de 200 días de edad a los que se suministró 0.2 ml de aceite de girasol por día se dividieron en los siguientes grupos: (1) dosis diaria de 10 nmol/g de oleoil-estrona; (2) restringido; (3) control. Las variables medidas fueron: proteínas corporales totales, agua y lípidos; pesos de los panículos adiposos retroperitoneal y epididimario; urea, glucosa, insulina, colesterol y triacilgliceroles plasmáticos. Los valores de biomasa y de ingesta de alimentos se registraron diariamente. Los grupos tratados con oleoil-estrona y restringido mostraron variaciones similares en composición corporal y disminuciones significativas en peso corporal debidas a reducciones en la ingesta de alimentos. Los tratamientos con oleoil-estrona y restringido disminuyeron significativamente los pesos de los panículos adiposos retroperitoneales, pero no de los epididimarios. En los grupos tratados con oleoil-estrona y restringido la hiperglicemia disminuyó y la insulinemia lo hizo en forma significativa. Los valores de colesterol total plasmático normal y la hipertriacilglicerolemia característicos de las ratas Beta disminuyeron fuertemente comparados con los controles, aunque alcanzaron valores significativamente diferentes entre los grupos tratados con oleoil-estrona y restringido. El colesterol total plasmático aparece como más sensible a la restricción calórica que los triacilgliceroles a través de un efecto específico mediado por la oleoil-estrona.

Palabras clave: Oleoil-estrona; Ratas Beta endocriadas; Obesidad; Pérdida de peso; Diabetes.

     Oleoyl-estrone (OE) has been reported as one fatty acyl ester of estrone carried by lipoproteins with an important function in the signaling system of energy homeostasis¹. It has been suggested that OE does not act through leptin, but that these hormones share a close functional relationship². Chronic administration of OE either via i.v. in liposomes or orally, to lean or obese rodents, results in a progressive loss of fat stores due mainly to lower caloric intake while energy expenditure is maintained³. Rodents on OE treatment lose weight without any apparent side effect³. It has been demonstrated that the presence of OE in the diet promotes a loss of appetite not directly mediated by NPY (neuropeptide Y)⁴ nor by CRH (corticotropin releasing hormone)⁵ without taste aversion side effects². OE  produces alterations in the plasmatic lipidic profile in both lean and obese rats (lowered plasma total cholesterol as well as other types of lipids in rats)⁷. Moreover, in OE treated rats variations of internal (e.g. retroperitoneal) white adipose tissue (WAT) depots were larger than those of external (e.g. epididymal) ones⁸. Treatment with OE has been described  to affect body protein less intensively than hypocaloric dieting alone (6) but no comparison with pair-fed animals has been reported. 
     Inbred IIM Beta (Beta) strain of rats, from the IIM stock^9-11, is a suitable model for human hypertriacylglycerolemic mild obesity and progressive glucose intolerance evolving towards type 2 diabetes¹².
     The aim of this study was to describe the effects of OE on spontaneously obese and diabetic Beta rats as well as to determine OE effects dependent on the caloric restriction it promotes by comparing OE treated rats to pair-fed ones.

Materials and Methods

Rats, feeding and oleoyl-estrone

IIM Beta (Beta) strain of rats form IIM stock^9-11 has been inbred by a non-regular system using small populations. Twenty five 200 day-old male Beta rats with well developed obesity and diabetes, were allocated in individual cages four days before the beginning of the treatments. They were kept under standard lighting (12h light-12h dark), temperature (21 ± 2° C) and relative humidity (60-75%), and were fed standard rodent chow pellets (Cargill^®). The composition of this feed in g/100 g was: moisture: 10.9; crude protein (f: 6.25) 23.9; lipids: 5.5; fiber: 8.2; digestible carbohydrates: 45.2; minerals: 6.3. The energy equivalence, after correcting for digestibility was 1335.9 kJ/100g.  The mean ad libitum daily intake before the beginning of the treatments was 28 ± 4g = 374 ± 53 kJ. Additional 7.5 kJ from  the sunflower oil given by stomach probe were daily added during the experiment.
     Oleoyl-estrone was synthesized from oleoyl-chloride and estrone in an anhydrous pyridine medium by M. Alemany (Departament de Bioquimica I Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain)³.

Experimental design

Three groups of Beta 200 days-old male rats were randomly selected for the following treatments:1- (OE): fed ad libitum plus an oral daily dose of 10 nmol/g  body weight of oleoyl-estrone (n=10). 2- (PF): pair-fed to OE (n=7). This group was offered the average of the daily amount of feed eaten by the OE group, thus resulting in an imposed food restriction. 3- (CO): controls, fed ad libitum (n=8) (Fig. 1).

Fig. 1.–  Percentage of daily food intake (mean ± SEM) in Beta 200 days old male rats with mild obesity and type 2 diabetes during 10 days on daily treatment with oleoyl-estrone (OE) (n=10); pair-fed (PF) (n=7) and controls (n=8). One hundred percent corresponds to the daily intake over 4 days previous to treatments.

     All the rats received a daily oral gavage of 0.2 ml sunflower oil, with oleoyl-estrone (OE), or without it (PF and CO), during 10 days. Body weight and food intake were recorded daily.
     Rats were maintained and handled following the guidelines established by the Bioethics Committee of Facultad de Ciencias Medicas, Universidad Nacional de Rosario, Rosario, Argentina.

Body composition analyses

Rats were sacrificed by ether overdose on day 11 after the beginning of treatments and blood was collected by partial decapitation. Hair and gastrointestinal content were removed. Epididymal and retroperitoneal fat pads were dissected, weighed and restored to the rest of the carcass which included skin and subcutaneous fat depot. This was weighed and stored at –20 °C until processing. The frozen carcass was crushed and minced until homogenized. Duplicate aliquots from every rat were used for the following analysis:
     Moisture: estimated by drying at 102-105 °C until weight loss in 1 h < 1mg. (AOAC 1990, method 950.46)¹³.
     Fat content: determined on the dried samples with  chloroform/methanol 50/50 (Folch extractant) in a discontinuous Soxhlet extractor¹⁴.
     Crude protein: calculated from Kjeldahl method. (AOAC 1990, method  984.13). The factor used to express protein content from nitrogen concentration was 5.5¹⁵.
     All trials were carried out in duplicate.
     Body composition data in relation to actual body weight changes were used to estimate the variations (gain or loss) in protein, water and lipid at the end of the treatments. All rats on day0 were assumed to share the same body composition: % of body components for all the rats on day 0 were assumed equivalent to those of controls on day 11, assuming the lack of variation over ten days in this adult age.

Plasma analyses

Glycemia: determined by an enzymatic method using a commercial kit (Wiener Lab, Argentina)¹⁶.
     Uremia: determined by a specific enzymatic method for blood and urine urea determination with a commercial kit (Wiener Lab, Argentina)¹⁷.
     Cholesterolemia: cholesterol was determined by an enzymatic method using a commercial kit (Wiener Lab, Argentina)¹⁸.
     Triacylglycerolemia: determined by an enzymatic Trinder colorimetric method for serum or plasma triacylglycerols determination with a commercial kit (Wiener Lab, Argentina)¹⁹.
     Insulinemia: quantified by a solid-phase I¹²⁵ radioimmunoassay designed for the quantitative measurement of insulin in serum using a commercial kit (Coat-A-Count Insulin-Diagnostic Products Corporation, USA)²⁰.

Statistics

Data are presented as means ± standard error (SEM) and have been compared using ANOVA and two-tailed t test for unpaired data. Mean values were considered significantly different for p < 0.05.

Results

     OE promoted a reduction in food intake. The largest reductions in OE as well as in PF were from day 4  to day 7 (Fig. 1). Total food –and caloric intake– were significantly higher in CO: 278 ± 14g  (3713.8 ± 187 kJ) compared with OE: 155 ± 10g (2070.6 ± 134 kJ) or PF: 149 ± 5 g (1990.5 ± 67 kJ) (p < 0.001).
     The average initial weight of the rats was 436.28 ± 8.51 g. Treatment with OE as well as PF resulted in a body weight loss of about 10% on day 11, while CO gained about 2% of their initial biomass (Fig. 2).

 Fig. 2.–  Percentage of initial biomass (mean ± SEM) attained by 200 days old male Beta rats with mild genetic obesity and type 2 diabetes during 10 days on daily treatment with oleoyl-estrone (OE) (n=10), pair-fed (PF) (n=7) and controls (n=8). One hundred percent corresponds to the mean biomass  attained by each rat over 4 days previous to treatments.

     Table 1 summarizes the variations of biomass as well as the body amounts of protein, water and lipid following treatments. Amounts on day 0 were assumed alike for all the rats and estimated  equivalent to those of controls on day 11, assuming the lack of significant variation  in  this adult age. Actual variations of biomass and estimated variations of total protein, water and lipid in OE were similar to those in pair-fed group (Table 2).

TABLE 1.–  Biomass and body protein, water and lipid in 200 days-old male Beta  rats with mild obesity and type 2 diabetes treated with oleoyl-estrone or pair feeding

¹(mean ± SEM). Biomass  day 0 vs day11: OE (p = 0.0005); PF (p< 0.0001); CO (p > 0.05); ²(mean)Estimations of body protein, water and lipid on day 0 = g/100g of CO on day 11, assuming the lack of variation in body composition over 10 days in these adult rats; ³(mean) estimation of body protein, water and lipid on day 0 (g); ⁴(mean ± SEM) Body protein, water and lipid on day 11 (g/100g); ⁵(mean ± SEM) body protein, water and lipid on day 11 (g). Values sharing one superscript letter horizontally are not significantly different (p>0.05).

TABLE 2.– Variations of biomass and body components in 200 days-old male Beta rats with mild obesity and type 2 diabetes treated with oleoyl-estrone or pair feeding

¹Variations of biomass (mean ± SEM); and  ²estimated variations of body components (mean). Estimated variations of body protein, water and lipid = g on day 11 – g estimated on day 0. (Grams on day 0 were estimated from biomass on day 0 and % of body components of controls on day 11, assuming the lack of variation in body composition over ten days in these adult rats). Values sharing superscript letter are not significantly different (p>0.05).

     Compared to PF rats, OE treatment resulted in higher plasma triacylglycerols and lower total cholesterol, while CO plasma lipid values were both significantly higher than either treated groups (Table 3). Plasma glucose and urea in OE, though lower than CO, did not differ significantly. Plasma insulin levels in CO were the highest: CO vs PF (p = 0.0120) and CO vs OE (p = 0.0114) (Table 3).

TABLE 3.– Plasma parameters in 200 days-old male Beta rats with mild obesity and type 2 diabetes treated with oleoyl-estrone or pair-feeding

     Relative weights of epididymal fat depots (g/g of total body weight x 100) were alike in the three groups, CO: 2.05 ± 0.17, OE: 2.04 ± 0.11 and PF: 2.03 ± 0.12 (ns). On the contrary, retroperitoneal fat depot relative weights were CO: 2.65 ± 0.14, OE: 2.12 ± 0.17, PF: 1.90 ± 0.10.  CO was significantly higher than OE and PF  (p = 0.02), while OE was not significantly higher than PF.
     Though not tested yet, the activity and locomotion of the OE treated rats were evidently lower than those of controls. This unusual behaviour was transient, as the rats were apparently recovered at about day 8 of treatment. On the other hand, the activity and exploratory behaviour of pair-fed rats were indistinguishable from those of controls, without any evidence of stress derived from lower feeding.

Discussion

Beta strain of rats is a suitable model for human hypertryacilglycerolemic mild obesity and progressive glucose intolerance evolving towards type 2 diabetes¹². Food intake was  significantly reduced in Beta obese rats on oleoyl-estrone treatment and of course in pair-fed counterparts. Consequently, a significant reduction in biomass compared to controls took place after 10 days on either OE or PF treatments. Body weight losses were basically from fat, accompanied by losses of water and protein in a much lower proportion. The reduction in total lipid content in OE group resulted similar to that in PF one. However, OE retroperitoneal fat depots tended to remain heavier than those in PF group, suggesting an OE specific-mediated reduction perhaps in a type of WAT depot different from those studied here. This might be mesenteric fat, since its decrease was the highest in Zucker lean rats on OE treatment⁸. The unaltered epididymal fat depot weight suggests that the maintenance of this WAT site, associated with the species preservation, is beyond the influence of the OE dose given or the level of caloric restriction achieved in these experiments.
     Plasma parameters for controls were typical for Beta mature males: hypertriacylglycerolemia, normal plasma total cholesterolemia and high glycemia baseline values. Both OE and PF showed lower plasma total cholesterol and triaclyglycerol levels than CO. Nevertheless, these values were  significantly different between OE and PF, accounting for a specific OE-mediated reduction in both types of lipids. Surprisingly, plasma triaclyglycerol levels on OE treatment were less sensitive to the reduction of body weight than plasma total cholesterolemia.
     Reduction in plasma triacylglycerolemia in OE treated group was lower than in PF. This could be a consequence of the increased liberation of fatty acids from the fat depots.  Meanwhile, an enhanced lipoprotein turnover and lipid transport convey fatty acids to the liver, where an intense resynthesis of triacylglycerols by OE takes place⁷. The least triacylglycerols concentration was found in PF group, suggesting a fasting-like behaviour with high fatty acid oxidation, after its nonspecific mobilization from fat depots and activation of lipoprotein lipase. Plasma total cholesterol normal values of Beta rats were practically halved on OE treatment, suggesting an improved cholesterol handling that may be, at least in part, a consequence of the OE enhancement of lipoprotein turnover⁷. The marked reduction in plasmatic cholesterol levels indicates an alternative metabolism. As long as in these studied conditions there are no special cholesterol needs for growth or cell differentiation, the most probable cholesterol alternative metabolic sink would be the liver⁷, which takes up lipoprotein particles carrying cholesterol from the extrahepatic tissues and secreting cholesterol and bile acids from the body²¹. Cholesterolemia reduction in PF, though important, was of less magnitude.
     Although nitrogen balance was not performed, plasma urea within the physiological range in OE group might express the lack of amino acid mobilization. The reduction in the hyperglycemic level on OE treatment failed to reach the euglycemic range in these rats. The strong reduction  in insulin concentration could not be attributed to OE, but to reduced caloric intake, since it was observed with both treatments.
     In conclusion, for these Beta rats OE-mediated effects resulted similar to those described by Alemany et al^{1-3, 6-8} in various rodent strains. Some disagreements might be attributed to differences of genetic line, age (adult stabilized Beta rats vs younger ones) and /or sex.
     Though OE has been referred not to have any apparent side effects in the various rodent strains assayed, the unusual behaviour of Beta OE-treated rats would be worth pursuing in further investigations.

Acknowledgements: We are grateful to Dr Maria Alemany for kindly providing the oleoyl-estrone used in these experiments. We are indebted to Wiener Lab, Argentina, for providing the kits employed for glucose, cholesterol, triacylglycerols and urea analyses.

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Received: 12-12-2003
Accepted: 10-04-2004