Dietary sodium restriction exacerbates age-related changes in rat adipose tissue and liver lipogenesis☆
Section snippets
Materials and methods
Male Wistar rats weighing 35 to 40 g (just weaned) were fed for 1, 2, or 3 months with either an LSD (0.06% Na+) or a normal salt diet ( NSD) (0.5% Na+) (TD 92141 and TD 92140, respectively, Harlan Teklad, Madison, WI). Except for Na+ content, these 2 diets have the same composition and contain 25% protein, 51% carbohydrates, and 6% fat. Rats were housed in individual cages in a controlled-temperature environment (25°C ± 2°C) with a 12-hour light:dark cycle and water ad libitum. Food and water
In vivo lipogenesis measurements
3H2O (5 mCi) was injected intravenously through a Silastic catheter (Dow Corning, Midland, MI) inserted into the right jugular vein 2 days before the experiment, and the rats were killed by decapitation 60 minutes after label injection. The retroperitoneal fat depots and liver were rapidly removed and weighed. Total lipids from tissue samples were extracted with 2:1 chloroform:methanol by the procedure of Folch et al.10 3H2O not incorporated was removed from the inferior phase (predominantly
LPL activity
An anhydrous emulsion of tri-14C-oleoylglycerol, stabilized by lecithin, was prepared in glycerol as described by Nilsson-Ehle and Schotz.14 The assay substrate solution was prepared daily by adding 2 vol of the emulsion, 2 vol of Tris buffer 0.2 mol/L (pH 8.8) containing 6% (wt/vol) bovine serum albumin and 1 vol of 36-hour fasted rat serum. After vigorous shaking in a Vortex mixer for 5 seconds, the new emulsion was ready for immediate use. Retroperitoneal adipose tissue was homogenized in
BAT temperature response to norepinephrine injection
Animals were anesthetized with pentobarbital (40 mg/kg, intraperitoneal [IP]), and a catheter was inserted into the right jugular vein using a Silastic tubing (no. 602-135, Dow Corning). The BAT temperature was measured by making a small surgical incision above the BAT and placing a small thermistor between the 2 lobes of the tissue. Core (colonic) temperature was measured by inserting a similar thermistor 5 cm beyond the anus (256S; Dixtal Tecnologia, Ribeirão Preto, São Paulo, Brazil).
Guanosine diphosphate binding to BAT mitochondria
BAT mitochondria were isolated as described by Cannon and Lindberg17 and suspended in 0.25 mol/L sucrose to a final protein concentration of 8 to 10 g/L. Binding of purine nucleotide to isolated mitochondria was measured with 3H-guanosine diphosphate (GDP) (Amersham, Little Chalfont, UK) as described by Nicholls,18 except that 14C-inulin (NEN Products, Du Pont, Boston, MA) was used to correct for the amount of 3H-GDP in the trapped medium. Mitochondrial protein was determined by the method of
Other chemical analyses
Tissue lipid content was determined as described.10 After removal of the intestinal tract, the remaining carcass was weighed, autoclaved to soften the bones, homogenized in 500 to 1,000 mL water, and the lipid content was determined gravimetrically after extraction by the Folch procedure.10, 20 Plasma glucose was determined using a Beckman glucose analyzer (Fullerton, CA). Plasma TAG, free glycerol, and free fatty acid (FFA) levels were assayed enzymatically using commercial kits from Labtest
Statistical methods
Data are expressed as means ± SEM. Differences between means were analyzed using 2-way analysis of variance (ANOVA) or Student’s t test, as appropriate, with P < .05 as the criterion of significance.
Results
Figure 1A to D shows the results of measurements made in metabolic cages during the 10 days that preceded the terminus of each of the periods (1, 2, and 3 months) of diet consumption The data in Fig 1A and 1B show that, except for body weight values somewhat higher in LSD rats at the end of the second month, no significant difference was observed in body weight and food intake of LSD and NSD rats during the periods examined. Figure 1C and D show that, as expected, urinary volume and water
Discussion
Aside from the expected LSD-induced changes in urinary volume and water intake, the main findings of the present study were the alterations produced by the diet in the metabolism of adipose tissue. An adequate discussion of these alterations requires a previous analysis of the results obtained in the control rats, fed the NSD. In these animals, prolongation of the experimental period from 2 to 3 months resulted in a marked reduction in adipose tissue lipogenesis that was accompanied by
Acknowledgements
We thank Elza Aparecida Filippin, José Roberto de Oliveira, Neusa Maria Zanon Resano, and Victor Diaz Galbán for technical assistance.
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Supported by grants from Programa de Núcleos de Excelência (PRONEX, no.76.97.1030.00) and Conselho Nacional de Pesquisa (CNPq, no. 141042/1998-2).