Lactation greatly increases the nutrient requirements of rat dams. These are primarily met by an increase in their food intake and mobilization of body energy reserves. Nutrients are shifted to the mammary gland with certain priority. Tissue protein is not strictly considered to be a reserve, but its content in dam tissues changes noticeably during the pregnancy-lactation cycle. The protein content of carcasses from pregnant rats is greater than that from non-pregnant rats in order to subsidize fetal growth. Since the early studies by Spray, several authors have demonstrated a net loss of body protein during lactation in well-nourished mice and rats; additionally, Naismith et al. and Moore and Brasel reported the loss of fat mass in lactating rats. Furthermore, muscle protein turnover has been found to be increased during lactation. Nevertheless, some studies have reported that the carcass protein does not change during lactation, either in rats or in mice. Protein loss from maternal tissues could be explained by intracellular protein hydrolysis, where lysosomal enzymes must be involved. Cathepsins are the proteolytic lysosomal enzymes, and cathepsin D, the most studied of these enzymes. This investigation was designed to study the changes in cathepsin D activity in the liver, muscle and mammary gland throughout lactation and weaning as an indicator of intracellular proteolytic activity.

Materials and Methods

Sucrose, Tris-HCl, hemoglobin, Triton X-100, pepstatin and Folin-Ciocalteu reagent were obtained from Sigma Chemical Co. (St. Louis, MO, USA). All other chemicals were reagent grade.

Animals and Protocol

Female Sprague-Dawley rats, 16 weeks old, weighing 240–280 g, were housed at 22–24°C and 12-h light-dark cycles (7:00 a.m. lights on). They had free access to water and to a commercial nonpurified diet containing 23% protein, 4.5% crude fat, 6% crude fiber (Laboratory Rodent Diet, PMI Feeds Inc., St. Louis, MO, USA). Rats were mated for 7 days with males of the same strain. The day that spermatozoa were identified by routine vaginal smear (9:00 a.m.) was considered to be the first day of pregnancy. Rats were then caged individually. At parturition (designated as day 1 of lactation), litters were adjusted to eight pups. Rat dams were randomly assigned to any of six groups of 14 animals each. Animals were killed on days 14 and 21 of lactation, representing the maximal milk production and final stages of lactation. Two groups were slaughtered without litter separation and were designated L-14 and L-21. Two other groups were killed 24 h after removal of their litters and were designated W-14 and W-21. Two age-paired groups of nonpregnant, nonlactating rats were killed once they reached an age equivalent to that of L-14 or L-21 rats. Slaughterings were done between 9:00 and 10:00 a.m. in order to avoid the effects of potential circadian variations.

Procedures

After killing the animals by cervical dislocation, the liver, mammary tissue from pectoral, abdominal and inguinal regions of both sides, and the whole muscle package of the right hind limb were removed and kept in an ice-cold 0.25 M sucrose solution, buffered with 0.01 M Tris, at pH 7.5. Connective tissue and fat were grossly removed. Tissues were then homogenized (Tissumizer, Tekmar Co., Cincinnati, OH, USA) in the buffered solution described above (1:5 w/v). Homogenates were used as the source of the enzyme for enzyme assays. Protein concentrations were determined as described by Itzhaki and Gill, using bovine serum albumin as standard.

Cathepsin D assay

Cathepsin D activity was measured by the method described by Iodice et al.. The final assay conditions were as follows: 1 ml of incubation mixture contained 0.1 M formate buffer, pH 3.5, 2% acid-denatured hemoglobin, 0.2% Triton X-100, and 100 ?l of the homogenates. Samples were incubated in triplicate for 60 min at 45°C. The reaction was stopped by the addition of 5 ml of 3% trichloroacetic acid. Tubes were centrifuged at 3500 g for 20 min (model J-6B, Beckman Instruments, Inc., Fullerton, CA, USA). Enzyme activity was measured in an aliquot of the supernatant by adding Folin-Ciocalteu reagent and reading its absorbance at 700 nm. Enzyme activity was completely inhibited with 1.6 ?g of pepstatin.

Results were referred to a standard curve of tyrosine (0–40 ?g), so that cathepsin D specific activity is expressed as micrograms of tyrosine released per milligram of protein during 1 h of incubation.

Results

Liver

Specific activity of cathepsin D at the peak of milk production (L-14) and at the end of lactation (L-21) was 50 and 42% higher, respectively (p <0.001) than in control rats V-14 and V-21. Litter separation for 24 h (W-14 and W-21) was followed by a sharp return of cathepsin D activity close to control values.

Mammary gland

The specific activity of cathepsin D in tissue from rats at L-14 and at L-21 was 164 and 204% higher, respectively, than in controls (p <0.001). Litter separation resulted in a decrease of the enzyme activity in W-14 and W-21; however, this reduction was only significant at W-14. In both cases, the activity remained significantly greater than in controls .

Muscle

Cathepsin D activity in muscle was the lowest among all studied tissues. No significant changes were observed throughout lactation or after removal of litters.