Effect of dietary calcium (Ca) on body composition and Ca metabolism during growth in genetically obese (β) male rats.

MAROTTE CLARISA; WEISSTAUB ADRIANA; BRYK GABRIEL; OLGUÍN MARÍA C; POSADAS MARTA; LUCERO DIEGO M; SCHREIER LAURA; PITA MARTÍN DE PORTELA MARÍA L; ZENI SUSANA N

EUROPEAN JOURNAL OF NUTRITION

DR DIETRICH STEINKOPFF VERLAG

Año: 2013 vol. 52 p. 297 - 305

1436-6207

Dietary calcium (Ca) and body composition and Ca metabolism during growth, in genetically obese (β) male rats. Marotte Clarisa1,2, Weisstaub Adriana3, Bryk Gabriel2, Olguin Maria C4, Posadas Marta4, Lucero Diego M5, Schreier Laura5, Pita Martín de Portela Maria L3, Zeni Susana N1,2,* 1National Council for Scientific and Technological Research (CONICET); 2Medical Osteopathies Section, Clinical Hospital, Buenos Aires University (UBA); 3 Food Science and Nutritional Department, School of Pharmacy and Biochemistry (UBA); 4 School of Biochemistry and Pharmacy, Rosario National University (UNR); 5Lipids and Lipoproteins Laboratory, Clinical Biochemistry Dept. (UBA). *Corresponding author: Susana N Zeni Cordoba 2351-8vo. Piso (1120) Buenos Aires Argentina Tel-FAX: 541159508972 snzeni@hotmail.com Short page heading: Calcium intake and body composition interrelationship in obese rats. Word count of text: abstract: number of references: 33; number of tables: 4 and figures: 4 Conflict of interest: None Abstract Obese  rats could be a suitable model to evaluate calcium intake (CaI) and obesity interrelation during growth. Objective: The present study comparatively evaluated Ca absorption and retention, and changes in body composition in spontaneously genetically obese (β) male rats fed three different dietary Ca levels: 0.9% (HCa); normal: 0.5% (NCa); low: 0.2% (LCa). Methods: During pregnancy female rats fed isocaloric diets only varied in Ca content. Male pups continued feeding the same maternal diets up to 60 days. Apparent Ca absorption percentage (CaA %), Ca balance (CaB), body composition, glucose, triglycerides (TGL) and insulin levels were evaluated. Results: Food consumption and body weight (BW) were higher in LCa group vs. NCa and HCa groups (p<0.01) which did not present differences between them. LCa group presented the highest body fat, liver weight, perigonadal and retroperitoneal fat (p<0.05), conversely body ashes and total skeleton bone mineral content were significantly lower than both, NCa (p<0.01) and HCa groups (p<0.01). CaB (mg/d) vs CaI (mg/d) reached a plateau with the highest CaI (r=0.985, p<0.001). CaA%, serum glucose, insulin and TGL levels rose as CaI decreased (p<0.01). Conclusions: Although future studies are required, a low Ca consumption in this strain of rats could modulate BW inducing changes in several lipid metabolism parameters that lead to an increase in body fat. Keywords: Low calcium diet- bone mass- fat Introduction Calcium (Ca) is an essential mineral to achieve an optimum peak of bone mass during growth and development and to maintain skeletal integrity in the adult life. More than 99% of total body Ca is in the skeleton where it is associated with phosphate to form the apatite crystal. The remaining Ca is present in soft tissues, where ionic Ca (Ca++) is involved in a great number of physiological functions such as nerve excitability regulation, muscle contraction and blood coagulation, among others. In 2004, Zemel MB et al [1] suggested that Ca is also involved in the modulation of the energetic metabolism, exerting an ?anti-obesity? effect which was supported later by several researches from this group both, in humans and in mice [2-4]. This theory would connect two worldwide growing problems: the well known high prevalence of low Ca intake (CaI), and the increment in human overweight and/or obesity. Several studies have shown that adiposity and CaI are inversely correlated, especially during growth [5-8]. In addition, epidemiological observations from the National Health and Nutrition Examination Survey III (NHANES III) [2], the Coronary Artery Risk Development in Young Adults (CARDIA) study [5, 7] as well as the post-analysis of the data from the National Nutrition and Health Survey (ENNyS) conducted in Argentina during 2004-2006 [10] evidenced an inverse relation between both, overweight/ obesity, and CaI. The effect of different dietary Ca content on bone metabolism was extensively studied in normal rats; however, according to our knowledge, there are no studies about this effect in an inbred strain of rats that develop spontaneous obesity during the prepuberal period, as the IIMBβ strain [9]. Moreover, this strain of rats may be considered as a suitable model to evaluate CaI and body composition interrelation, an controversial subject until now. On these bases, the aim of the present study was to evaluate Ca absorption and retention, and changes in body composition in spontaneously obese rats feeding three different dietary Ca levels (low, normal, high). 1.Materials and Methods 1.1Animals: Female IIMbb rats (250-300g) were housed at controlled room temperature (21 ± 1°C) with 55 ± 10% humidity under 12-h light/dark cycles. The IIMb/β rats develop obesity and type II diabetes from puberty onwards. They were obtained by genetically-environmental maladjustment and a high inbreeding degree. These obese rats also develop hypertryacilglyceridemia without hypercholesterolemia with a progression from glucose intolerance to type II diabetes and hypertension associated to obesity [11]. Throughout the experimental period, rats were allowed to access deionized water and food ad libitum. They were maintained in keeping with the National Institute of Health Guide for the Care and Use of Laboratory Animals and the protocol was approved by the Bioethics Committees of the Universities of Buenos Aires and Rosario. 2.2 Experimental design: Adult female rats were mated by placing one male rat in a cage with four females. From pregnancy to the end of lactation dams were housed in individual stainless steel cages receiving one of three studied isocaloric diets, which only varied in the Ca content: Low (LCa), Normal (NCa): and High (HCa) (table 1) [12]. Within 24 hs after delivery the numbers of litters were adjusted to 8-9 per dam. At weaning, male pups (n=8 per diet) continued feeding their mother´s diets up to reach 60 days of age. Food consumption was recorded 3 times/week and body weight (BW) was recorded once a week and food efficiency (g/g) was calculated according to the following equation: Food efficiency = Food intake (g)/increase in BW (g) Blood was collected at the end of the experience (T=60) in as fasting state under anesthesia (0.1 mg ketamine hydrochloride /100g BW and 0.1mg acetopromazine maleate /100g BW). Then, animals were sacrificed by CO2 and intraperitoneal and retroperitoneal fat and liver were removed and weighed. 1.2Diets: The 3 experimental diets were prepared according to the American Institute of Nutrition Rodent Diets Recommendations settled in 1993 (AIN´93) [12]. Ca was provided by CO3Ca (Analytical grade, Anedra, Argentina) to supply the 3 different Ca contents: HCa: 0.9% Ca; NCa: 0.5% Ca; LCa: 0.2% Ca. Composition of diets is outlined in Table 1. 2.3 Balance and apparent Ca absorption: During the last 3 days of the experience the animals were individually lodged in plastic metabolic cages and food consumption, feces (F) and urine (U) samples were collected to calculate Ca absorption (CaA) and Ca balance (CaB) [13]. Apparent CaA, as percentage of CaI (CaA %) and CaB (mg) were calculated according to the following equations: CaA % = (Ca I ? Ca F/ Ca I) x100 and CaB = (Ca I ? Ca F ? CaU) 2.4 Densitometry: At the end of the experience and before sacrifice, total skeleton bone mineral content (BMC) was determined ?in vivo? under light anesthesia with a total body scanner by dual energy x-ray absorptiometry (DXA) provided with a specifically designed software for small animals (DPX Alpha, Small Animal Software, Lunar Radiation Corp. Madison WI) as previously described [14]. In brief, all rats were scanned using an identical scan procedure. Precision was assessed by measuring one rat five times with repositioning between scans on the same and on different days. The BMC coefficient of variation (CV) was 3.0%. 2.5 Analytical Procedures: Body composition was determined according to the Association of Official Analytical Chemists (AOAC) methods [15]. Water was measured by desiccation in an oven at 1002C until constant weight. Total body fat content was determined on the dried ground carcasses, with petroleum ether in a Soxhlet extractor. White and crystalline ashes were obtained at 550C. Nitrogen (N) was determined by Kjeldahl method and the percentage of protein content was calculated as N x 6.25 (% protein = % N x 6,25) [16]. Feces were dried in an oven at 42 º C during 12hs, ground to 0.5 - 1.0 mm mesh and dried again at 105º C during 2 hours. Dried fecal samples (1.5 -2.0 g) were homogenized in a Potter Elvejahn device and lipids were extracted with chloroform/methanol (2:1 v/v). Total lipids were determined gravimetrically after solvents evaporation. Defatted feces were dried under an IR lamp and powdered in a processing machine supplied with a titanium blade. Feces and diets were digested with nitric acid, in a microwave using Parr bombs to determine Ca and phosphorus (P) contents [17]. Ca concentration in diets, feces, urine and ashes was determined by atomic absorption spectrophotometer [18]. Lanthanum chloride (6500 mg/L in the final solution) was added to avoid interferences. P in ashes was evaluated according to Gomori method [19]. At the end of the experience (T=60), serum glucose, cholesterol and triglycerides (TGL) were determined by habitual enzymatic methods and insulin was determined by enzyme immunoassay (Rat/Mouse Insulin ELISA Kit, Millipore, Billerica, MA, USA) 2.6 Statistical methods Results were expressed as mean ± standard deviation (SD). Data were analyzed using one-way analysis of variance (ANOVA), and Bonferroni multiple comparisons test was performed when significant differences were encountered. Statistical analyses were performed using SPSS for Windows 11.0 (SPSS, Inc. Chicago, IL). A value of P below 0.05 (P<0.05) was considered significant. 2.Results Impact on body weight and composition: Food consumption during all the experience was significantly higher in the LCa group compared to the other two studied groups (p<0.01) which did not present differences between them (Table 2). Figure 1 shows the evolution of BW for the 3 studied groups throughout the experience. From weaning, the LCa group had a significantly higher BW than NCa and HCa groups (p<0.01) which did not present differences between them. Body composition expressed as percentage of BW and total content are shown in table 2. Water adjusted by BW did not present significant differences among the three studied groups. LCa group showed the highest significant content of total fat and fat adjusted by BW compared to both NCa and HCa groups (p<0.01). As a consequence of the high BW, LCa showed the highest total protein content (p<0.01) however the lowest protein adjusted by BW which only reached significance compared to the NCa group (p<0.01). Total ash content was significantly lower in LCa and HCa groups compared to NCa group (p<0.01); conversely, ash content adjusted by BW was related to the dietary Ca content with the significantly lowest value for the LCa (p<0.0001) while NCa and HCa groups did not present differences between them. A linear significant correlation (r= 0.78; p<0.012) was found when the individual points of total ash content were plotted vs. lean mass expressed according to the following equation: Lean mass = BW ? total fat content ? total water content ? total ashes (Figure 3) Impact on glucose and energy metabolism: Table 3 shows fat intake, fecal excretion and fat absorption percentage obtained during the last three days of experience. Food consumption did not present differences among the studied diets (22.9±4.7; 25.2±4.8 and 24.6±1.7 mg/d for LCa, NCa and HCa groups, respectively). As a consequence, fat intake did not present differences among the three studied groups; fecal fat excretion was significantly higher in HCa group compared to both, LCa and NCa groups (p<0.01), which did not present differences between them. However, fat absorption percentage did not present differences among the three studied groups. Adipose perigonadal and retroperitoneal pads, liver weight, and serum levels of glucose, insulin and TGL rose as CaI decreased (p<0.01) while cholesterol levels were significantly higher in the NCa group compared with both LCa and HCa groups (p<0.05) which did not present differences between them (Table 3). Impact on Ca absorption and retention: Because there were no differences in food intake during the period in which absorption was evaluated, the CaI was directly related to the dietary Ca content and, the LCa group had the lowest CaI (p<0.01) (Table 4). In agreement with the ash content, total body Ca and P contents did not present differences between LCa and HCa groups which were significantly lower than the NCa group (p<0.001) (Table 4). Body Ca and P contents adjusted by BW were significantly lower in the LCa group compared to both, NCa and HCa groups without differences between them. In spite of these values the Ca/P ratio showed no differences between LCa and NCa groups which were significantly lower than the HCa group (1.36±0.04 and 1.36 ±0.03 vs. 1.54±0.05, respectively; p<0.05). Fecal and urinary Ca excretion during the period of absorption study was significantly related to the dietary Ca content (p<0.001 and p<0.01, respectively). CaB (mg) was directly correlated and CaA (% of CaI) was inversely correlated to the Ca content in the diets (Table 4). Indeed, CaB (mg/day) vs. CaI (mg/day) showed a positive function which reached a plateau in the high ranges of CaI (r=0.985, p<0.001) (Figure 4). The CaA% was significantly lower in HCa group as compared to LCa and NCa (p<0.01), which did not present differences between them. Impact on densitometry: Total skeleton BMC normalized by BW (BMC/100g BW) was significantly lower in the LCa group as compared to both, NCa (p<0.01) and HCa (p<0.01) groups; in addition, HCa group reach a BMC/100g BW lower than the NCa group (p<0.01) (Table 4). 3.Discussion In addition to the well-established role of Ca in bone and mineral metabolisms, there has been considerable recent interest in the hypothesis that CaI modulates body fat stores. The inbred IIMb/beta line of rats may be an interesting and suitable model to study the interrelationship between obesity and dietary Ca intake during the growth period because they develop progressive obesity and type 2 diabetes from puberty onwards without requiring a high energy intake. Impact on body composition: The results of the present report strongly suggest that, as previously observed in wild type male mice [20], dietary Ca may have a role in modulating BW in spontaneously obese male IIMbb rats. According to the results obtained in the present report rats fed the low isocaloric Ca diet exhibited a significant increase in food intake, BW gain, fat pad mass, and a decrease in protein content per kg of BW. Even though the differences in BW were observed from weaning, they may have been present before during lactation. Indeed, the dams received the different studied diets from mating and it is known that pups begin to consume a mixed diet (milk plus solid) approximately during the last week before weaning [21]. Although there was a decrease in the amount of total fecal fat excretion in the LCa group, no differences in the percentage of fat absorption were observed. These results suggest that the effect of the low Ca diet in increasing body fat accumulation may likely be quite small, yet high enough for the increase to reach significant levels throughout the experience. In addition, rats fed the low Ca diet showed an increase in liver weight that actually reflects higher body fat content, evidenced by the increment in adipose perigonadal and retroperitoneal pads as well as by the sum of both expressed as a percentage of BW. This increment could be the result of the higher food consumption of the LCa group; however, it should be kept in mind that there were no differences in food efficiency, calculated as food intake/BW (g/g) ratio, among the three studied groups. It is important to take into account that even though the process of fat and lean mass accumulation may have similar energy costs, lean mass per kg is metabolically more active and requires greater energy utilization than fat mass [22]. Hence, the increase in lean body mass observed in normal and moderate high Ca diets may shift energy away from fat stores. Daily food intake would decrease if the food were unpalatable [23]. However, no aversion to food was observed in the present report since no differences in food intake were observed between rats fed the isocaloric diets, i.e. the diet supplying the recommended AIN 93 Ca levels (0.5%) and that supplying a moderate higher level (0.9%). In spite of this, rats fed the moderate Ca diet tended to show lower fat content and BW gain rate. A similar finding was observed by Zhang Q and Tordoff MG [23] in adult female Sprague-Dawley rats allowed choosing between pairs of normal-energy density diets with different Ca levels. The authors observed a higher food intake in rats fed the low Ca diet. The inverse relationship between CaI and BW gain remains somewhat controversial. Several mechanisms have been proposed as potentially contributing to, and partly explaining, the overall impact of dietary Ca on BW or body fat mass. One suggested hypothesis is that dietary Ca levels may affect appetite and food intake [24]. In this regard, mammals may have evolved to respond to specific dietary components that act as indicators of dietary availability, so that, when food is plentiful, a mineral present in the diet (e.g., Ca) acts as the signal to reduce body fat mass accumulation [24]. Conversely, as seen in the present report, the low level of dietary Ca during a high growth rate period may serve as an indicator to promote food intake thus protecting the body against such deficiency. Indeed, food intake was highest in the LCa group mainly during the first two weeks of the experience. Another proposed mechanism, is the decreased availability of energy when Ca and fat are provided jointly. The high CaI could increase fecal excretion of fat, presumably via the formation of insoluble Ca fatty acid soaps in the gut or by binding of bile acids, which impairs the formation of micelles [25]. The low excretion of fat observed in the LCa group did not modify fat absorption percentage, although it may have induced a slight increase in fat availability, which throughout the experience could have caused a rise in fat accrual. In addition, when fat is absorbed, it enters blood circulation in the form of intestinally derived TGL-rich lipoproteins, ie, chylomicrons [25]. Thus, if Ca partly inhibits fat absorption, a decrease in TGL could be expected. Conversely, TGL would increase with a diet supplying a low amount of Ca. Although our obese rats spontaneously develop hypertryacilglyceridemia, the highest levels of TGL were observed in the LCa group. Hyperinsulinism and insulin resistance are characteristic features of obesity consistent with a decrease in insulin receptor activity or post-receptor defects [26]. The increment in the insulin/glucose ratio reflects fat-induced insulin resistance wich, according to the severity of the obesity and the rise in hypertriglyceridemia, could contribute to a higher body fat synthesis and fat storage [26]. In the present study, in addition to exhibiting the highest levels of TGL, the LCa group also showed the highest fasting levels of glucose, insulin and insulin/glucose ratio. As mentioned above, the strain of obese rats used herein spontaneously develops hypertryacilglyceridemia and insulin resistance after puberty. Both the latter were found to increase concomitant to an increase in weight gain rate by decreasing the Ca content in the diet. The negative correlation between dietary Ca intake and changes in BW or lipid content found in the present study is in agreement with several [1,5, 20,27,28,29], though not all [23], previous reports analyzing the potential effect of Ca on body fatness. The proposed biologic mechanism was related to the energy loss through increased expression of uncoupling proteins or increased lipid oxidation [20]. In addition to the inhibition of basal lipolysis, the mechanism might involve stimulation of the expression and activity of the fatty acid synthetase (FAS) enzyme by a protein product of the agouti gene. The C-terminal portion of this protein presents a three dimensional structure that functions as a Ca++ channel, allowing its entry into to a variety of cells (including adipocytes), and maintaining the intracellular concentration of [Ca++]i constant [30,31]. The increment in [Ca++]i stimulates the expression and activity of FAS, and at the same time increases insulin secretion by the pancreas, resulting in excessive deposition of TGL [2, 20]. Impact on Ca and bone metabolism: Ca bioavailability depends not only on luminal Ca concentration but also on age. According to AIN´93, CaA of normal rats feeding the recommended dietary Ca levels reached the highest levels at weaning and decreased thereafter to reach the lowest values in adult life [30]. All the obese rats studied herein were of similar age and were at the end of the high growth rate period; thus, the level of Ca absorption must have been affected by the total amount of dietary Ca. When Ca absorption was expressed in mg/d, as total CaI minus total FCa, the low Ca group reached the lowest level and no differences were observed between the normal and high Ca groups. Nevertheless, the percentage of CaA was similar between the low and normal Ca diets, and as the amount of Ca available in the intestine increased, the %CaA decreased. Urinary Ca excretion was very low in all groups (0.7-1.3 mg/d), the amount of absorbed and retained Ca was therefore similar. This finding may partly explain the bone mass results observed in the LCa and HCa groups when compared to the NCa group. Indeed, the lowest supply of dietary Ca limited the level of total body ash content and total skeleton BMC normalized by BW observed in the LCa group. Conversely, the highest dietary Ca content increased the supply of Ca without improving Ca bioavailability because although ash content was similar, the BMC/BW ratio remained lower than in the NCa group. The latter findings suggest that a plateau value exists above which an increased CaI value does not seem to have any additional effect. The dietary P level, which was the same in the three experimental diets, may have been a limiting factor in reaching higher levels of bone mass in the HCa. P is not considered a nutritional problem. However a high Ca/P intake can affect P absorption [32]. Although the absorbed P was not evaluated, the highest total body Ca/P ratio observed in the HCa group contrasts with the observed low bone mass and it could compensate the relative P deficiency regarding the NCa group. Another important finding suggesting such imbalance in the dietary Ca/P ratio is the lowest value in total body protein content which suggests impairment in nitrogen retention. It can be thought that the results of the present study may explain the differences found by other researchers differences found by other researchers between the experimental results in rats/mice and the human epidemiological studies regarding a better antiobesity effect of dairy Ca than that provided by supplements [7,31,33]. One limitation of the present work is the relatively short duration study, which focuses on the interrelationship between low CaI and fat mass accrual in a growing rodent model. Further studies should be conducted to confirm whether the observed results can be extended to an adult rodent model. Another limitation is that P absorption, a parameter that could have clarified the findings related to bone mass in the HCa group, was not measured. 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