Enzymes of adenosine metabolism of sheep adipose tissue: changes in activity with season, pregnancy and lactation

Activities of enzymes involved in the synthesis and degradation of adenosine have been measured in samples of adipose tissue from unmated sheep at various times of the year between January and October and from pregnant and lactating sheep. 5'-Nucleotidase activity increased during the spring in unmated sheep but this increase was suppressed in lactating sheep. Neither adenosine kinase nor adenosine deaminase activities varied significantly between January and October in unmated sheep. Lactation resulted in a rise in adenosine deaminase activity and a small decrease in adenosine kinase activity. Pregnancy had no obvious effect on the activities of any of the three enzymes noted above. Changes in adipocyte mean cell volume and number per g tissue and the concentrations of DNA and protein of the tissue are also described. Results are discussed in relation to changes in the capacity for lipid synthesis and mobilization which occur in response to season, pregnancy and lactation in sheep.     

Enzyme and protein turnover in adipose tissue in pregnancy and lactation

1. The relative rates of synthesis of fatty acid synthetase and the pyruvate dehydrogenase complex were measured in adipose tissue in virgin, late pregnant and early lactating rats after injection of l-[2,3-³H]alanine. The relative rate of synthesis of fatty acid synthetase decreased approximately 4-fold between 2 days prepartum and 2 days postpartum. The relative rate of synthesis of the pyruvate dehydrogenase complex did not change. 2. The fractional rate of total adipose tissue protein synthesis was measured by constant infusion with l-[U-¹⁴C]tyrosine. Total protein synthesis did not differ in virgin and 2-day lactating rats. The half-life of adipose tissue protein in virginn rats determined by decay of ¹⁴C label from protein after injection of NaH¹⁴CO₃ was 86.9 ± 6.7 h. This is in close agreement witht the half-life (82.5 ± 20 h) calculated from the fractional rate of protein synthesis determined by the constant infusion method.     

Adaptation of the small intestine during pregnancy and lactation in the rat.

During pregnancy and lactation in the rat the small intestine in general and the mucosal epithelium in particular gain weight. The specific activities of sucrase, lactate dehydrogenase and succinate-tetrazolium reductase remain constant and those of glucose 6-phosphate dehydrogenase and isocitrate dehydrogenase increase. There is no evidence that the reported decrease in absorption per unit area or weight of mucosal epithelium during pregnancy and lactation is due to decreases in enzyme activities within the epithelium. The pattern of enzyme change shows that the response of the gut to the stimuli of pregnancy and lactation must be a complex one, possibly involving increases in the specific activities of some enzymes.     

Role of insulin receptors in the changing metabolism of adipose tissue during pregnancy and lactation in the rat.

Changes in the volume, the rates of fatty acid synthesis and synthesis of the glycerol moiety of acylglycerols, the activity of lipoprotein lipase, and the number and affinity of insulin receptors of adipocytes, and concentrations of serum insulin, prolactin and progesterone were determined in virgin rats and in rats at various stages of pregnancy and lactation. Changes in the metabolic activities of adipose tissue appeared to be synchronized and primarily comprised a marked decrease in anabolic activity around parturition. In contrast, the number of insulin receptors (Kd 1.5 nM) per adipocyte doubled during pregnancy before returning to normal values around parturition. It is postulated that the increase in the number of insulin receptors is an adaptation to counteract the effects of insulin-antagonistic hormones during pregnancy and that the decrease in the number of receptors is primarily responsible for the loss of anabolic activity around parturition.     

Apparent dissociation between sympathetic activity and brown adipose tissue thermogenesis during pregnancy and lactation in golden hamsters

Sympathetic activity has been assessed by measurements of noradrenaline turnover in brown adipose tissue and in the heart of golden hamsters during pregnancy and lactation. Noradrenaline turnover was not significantly altered in either tissue in pregnant or lactating hamsters, despite the atrophy of brown adipose tissue that occurs during reproduction. This suggests that sympathetic activity and brown adipose tissue thermogenesis are dissociated during pregnancy and lactation in golden hamsters. The results also indicate that the large increase in food intake lactation does not lead to a diet-induced stimulation of the sympathetic nervous system.     

Changes in brown adipose tissue lipoprotein lipase activity and lipogenesis rate during pregnancy and lactation in the rat

Brown fat lipoprotein lipase activity did not change in the first two weeks of pregnancy whereas it decreased on day 18 of gestation and was lower during late pregnancy and lactation. Fatty acid synthesis rate, measured in vivo with (3H)H2O, showed a progressive increase until day 18 of gestation followed by a decrease on day 20 of pregnancy and a reduced lipogenesis rate throughout lactation. The early reduction in the pathways of fatty acid uptake and synthesis in brown fat during the breeding cycle of the rat suggests the possibility that a decline in the substrate supply was a factor contributing to the reduced thermogenic activity of brown adipose tissue after parturition.     

Growth hormone alters metabolic effects and proteolysis of insulin in adipose tissue during lactation.

To study the regulation of lipogenesis in adipose tissue by insulin and growth hormone during lactation, tissue was biopsied from primiparous bovines at 30 days antepartum and 60 days postpartum. Tissue was cultured for 24 hr or 48 hr in M199 with acetate and glucose, with a change of medium at 24 hr. The three in vitro treatments were: insulin and hydrocortisone at 10 and 50 ng/ml, respectively (IH); IH + 10 ng/ml of growth hormone (G10); and IH + 100 ng/ml of growth hormone (G100). IH allowed lipogenesis rates from 50% to 85% of those in fresh tissue. Addition of 10 ng/ml of growth hormone reduced (P less than 0.05) lipogenesis; at 100 ng/ml, the effect was only slightly greater. The hypothesis that insulin and growth hormone could be degraded by bovine adipose tissue was tested. Adipose tissue cell-free extracts degraded 125I-labeled insulin, but did not degrade labeled growth hormone. The insulin protease activity was further characterized and had a pH optimum of 7.1, a maximum hydrolysis of approximately 70%, and a hydrated molecular mass of approximately 23,000 daltons. Insulin proteolysis was inhibited by specific insulin protease inhibitors and stimulated by disulfide reducing agents. Bovine growth hormone, prolactin, and histone inhibited (P less than 0.05) the proteolysis of insulin, while bovine serum albumin, egg albumin, trypsin inhibitor, and lysozyme did not. Adipose tissue from pregnant and lactating bovines was sensitive to insulin and growth hormone, and growth hormone may modulate activity of an insulin-specific protease.     

Metabolic adaptations in goat mammary tissue during pregnancy and lactation

Metabolic adaptations of goat mammary tissue during pregnancy and lactation were monitored in serial biopsies of the tissue. Changes in the synthetic capacity of secretory cells were studied by combining measurements of enzyme activities with short-term culture of mammary explants to measure lactose, casein and total protein synthesis. By these criteria, the main phase of mammary differentiation began in late pregnancy and was essentially complete by Week 5 of lactation, coinciding with the achievement of peak milk yield. While milk yield declined after Week 5, the activities of key enzymes expressed per mg DNA and the rates of lactose and casein synthesis in mammary explants were maintained over a considerable period. The results suggest that changes in the synthetic capacity of epithelial cells may account for much of the rise in milk yield in early lactation, but are not responsible for the declining phase of milk production characteristic of lactation in ruminants.     

Metabolic adaptations during lactogenesis. Lactose synthesis in rabbit mammary tissue during pregnancy and lactation.

Lactose biosynthesis and relevant enzymatic activity in rabbit mamma ry tissue during various stages of pregnancy and lactation are investigated by using a tissue-slice incubation method in order to understand the temporal relationships. Ovulation was induced in 27 New Zealand white rabbits and they were bred by artificial insemination. Sacrifice occurred on days 15, 24, and 29 of pregnancy, and day 2, 5, 8, 15, and 22 post partum. Nucleic acids were extracted and concentratons of DNA determined spectrophotometrically at 600 nm with diphenylamine reagent and RNA determined with orcinal reagent. The tissue incubations were made with (U-14C) glucose. (14C) lactose was then separated by paper chromatography from unchanged radioactive glucose. Enzyme analysis including determining the activities of phosphoglucomutase, UDP-glucose pyrophosphorylase, and UDP-glucose 4-epimerase. Lactose synthase was determined, as well as, hexokinase. A biphasic adaptation in the rate of lactose synthesis and in the RNA concentration was noted during lactogenesis. The 1st increase in the rate of lactose biosynthes is occurred between days 15 and 24 of pregnancy. A 2nd substantial increase was noted immediately post partum. The overall rate of lactose biosynthesis increased 12-fold from day 24 of pregnancy to day 15 of lactation post partum, and then decreased from 15 to 22 days post partum. The RNA concentration/g wet weight of tissue and the ratio of RNA/DNA closely represented the biphasic ability of the mammary-tissue slice to synthesize lactose. Increases in the activities of UDP-glucose 4-epimerase and lactose synthase were most closely correlated with increases in the rate of lactose biosynthesis. UDP-glucose pyrophosphor ylase activity was unrelated with the ability to synthesize lactose, and hexokinase and phosphoglucomutase activities were variable during pregnancy and lactation. Lactose synthase activity was present by day 15 of pregnancy, but the ability to synthesize lactose was undetected until day 24 of pregnancy.     

Calmodulin content of rat mammary tissue and isolated cells during pregnancy and lactation.

The calmodulin content of heat-treated extracts of rat mammary tissue and isolated cells was measured by using stimulation of cyclic nucleotide phosphodiesterase (PDE) activity and radioimmunoassay (r.i.a.) procedures. The calmodulin content of mammary tissue increased 2.5-fold near the time of parturition, remained at the elevated level during lactation, then, after the onset of involution, decreased to values similar to those measured from mammary tissue of pregnant rats. When tissue from 15 animals in different stages of pregnancy, lactation and involution were compared, the r.i.a. gave 2.6-fold higher results than the PDE assay. To investigate further the increase in calmodulin content of mammary tissue, secretory and myoepithelial cells were enzymically dissociated from rat mammary tissue during different stages of pregnancy, lactation and involution. Protein, DNA, lactose, glucose-6-phosphate dehydrogenase and alkaline phosphatase were assayed to characterize the cell fractions. By using r.i.a., the calmodulin content per mg of protein in isolated secretory-cell fractions was high near parturition, then decreased and remained relatively constant during lactation. The amount of calmodulin expressed per mg of DNA in secretory cells did not show a marked change near parturition, suggesting a constant amount of calmodulin per cell. The calmodulin content of myoepithelial cells dissociated from mammary tissue and measured by using r.i.a. was 6-fold lower than in secretory cells and remained relatively constant during the course of lactation. The changing levels of calmodulin in rat mammary tissue during development are suggested to be related to proliferation and destruction of secretory epithelial cells, events that occur near parturition and involution respectively.     

Metabolism of adipose tissue in the fat tail of the sheep in vivo

The metabolism of the large mass of adipose tissue constituting the fat tail of the Syrian sheep has been investigated by measuring arteriovenous concentration (A-V) differences. The tail in situ in the intact anesthetized animal, as well as the isolated tail perfused with blood through a constant flow pump oxygenator, was used. In fed animals, the adipose tissue took up glucose and ketone bodies and released lactate and free fatty acids (FFA), although in some animals uptake of FFA also occurred. After 48-144 hr of fasting, uptake of glucose and ketone bodies continued and the FFA release increased. Total lipid esters and phospholipids were not released even after food had been withheld for 6 days. Insulin increased the A-V difference and the uptake of glucose, and reduced the FFA release. Adrenaline increased the A-V difference and uptake of glucose; the simultaneous increase in serum FFA was not accompanied by an increase in A-V difference for FFA in most experiments, which suggests that this adipose tissue is relatively insensitive to the lipolytic effect of the hormone. The effect of noradrenaline was similar to that of adrenaline. Glucagon hyperglycemia was not accompanied by increase in glucose uptake in most experiments.     

Metabolic adaptations during lactogenesis. Fatty acid synthesis in rabbit mammary tissue during pregnancy and lactation

1. Mammary tissue was obtained from rabbits at various stages of pregnancy and lactation and used for tissue-slice incubations (to measure the rate of fatty acid synthesis and CO(2) production) and to determine relevant enzymic activities. A biphasic adaptation in fatty acid synthetic capacity during lactogenesis was noted. 2. The first lactogenic response occurred between day 15 and 24 of pregnancy. Over this period fatty acid synthesis (from acetate) increased 14-fold and the proportions of fatty acids synthesized changed to those characteristic of milk fat (77-86% as C(8:0)+C(10:0) acids). 3. The second lactogenic response occurred post partum as indicated by increased rates of fatty acid synthesis and CO(2) production (from acetate and glucose) and increased enzymic activities. 4. Major increases in enzymic activities between mid-pregnancy and lactation were noted for ATP citrate lyase (EC 4.1.3.8), acetyl-CoA synthetase (EC 6.2.1.1), acetyl-CoA carboxylase (EC 6.4.1.2), fatty acid synthetase, glucose 6-phosphate dehydrogenase (EC 1.1.1.49), and 6-phosphogluconate dehydrogenase (EC 1.1.1.44). Smaller increases in activity occurred with glycerol 3-phosphate dehydrogenase (EC 1.1.1.8) and NADP(+)-isocitrate dehydrogenase (EC 1.1.1.42) and the activity of NADP(+)-malate dehydrogenase (EC 1.1.1.40) was negligible at all periods tested. 5. During pregnancy and lactation there was a close temporal relationship between fatty acid synthetic capacity and the activities of ATP citrate lyase (r=0.94) and acetyl-CoA carboxylase (r=0.90).     

Functional atrophy of brown adipose tissue during lactation in mice. Effects of lactation and weaning on mitochondrial GDP binding and uncoupling protein.

The thermogenic activity and capacity of brown adipose tissue were determined in mice during lactation and after weaning. There was a marked fall during lactation in the mitochondrial content of the tissue, and in GDP binding and the specific mitochondrial concentration of uncoupling protein. The lactation-induced functional atrophy of brown adipose tissue was fully reversible after weaning; mitochondrial content and the mitochondrial concentration of uncoupling protein were both restored, although GDP binding was not normalized.