Adaptations of adipose tissue metabolism and number of insulin receptors in pregnant sheep

The endocrine control of adipose tissue metabolism during pregnancy in sheep has been investigated. The number of insulin receptors of sheep adipocytes was increased during pregnancy. There was no apparent change in the concentration of serum insulin during pregnancy in sheep while the rise in serum progesterone concentration was smaller and more gradual than in rats. Net lipid deposition in adipocytes occurred during the first 55 days of pregnancy, probably due primarily to increased lipoprotein lipase activity. Net deposition of lipid had ceased by mid-pregnancy while by 125 days of pregnancy, the rate of fatty acid synthesis in adipose tissue was decreased and the serum fatty acid concentration had risen, suggesting the onset of net lipid mobilization in the tissue. Results are compared with those from other studies with rats; it would appear that different mechanisms regulate lipid deposition during pregnancy in sheep and rats.     

Changes in protein turnover during gestation in the foetuses,placentas, liver,muscle and whole body of rats given a low-protein diet

(1)Protein synthesis and content have been studied in skeletal muscle, liver, foetuses and placentas of pregnant rats given a protein-deficient diet. Changes which occurred during the anabolic and subsequent catabolic phases of pregnancy are compared with those in well-fed pregnant and in protein-deficient non-pregnant rats. (2) The normal increase in liver protein did not occur during pregnancy in the protein-deficient group. (3) Protein deficiency affected protein content of the placenta earlier and more severely than that of the foetus. (4) Rates of protein synthesis in liver, placentas and foetuses were enhanced above control values by protein deficiency. (5)_Muscle protein increased normally during the anabolic phase of pregnancy but fell during the catabolic phase, unlike values for weel-fed animals. (6) Muscle protein synthesis rates rose by similar amounts in well-fed and protein-deficient animals during the anabolic phase of pregnancy. The fall to starting values during the catabolic phase was sharper and earlier in protein-deficient animals, which could reduce demands on the body amino acid pool by an amount equivalent to over 50% of the needs for protein deposition in foetuses and placentas. Thus, changes in muscle protein synthesis in both anabolic and catabolic phases of pregnancy may afford some protection to foetal protein synthesis.     

Changes and localization of ovarian carbonyl reductase during pseudopregnancy and pregnancy in rats

The present study investigates changes in the activity and enzyme content of ovarian carbonyl reductase (CR), which catalyzes the reduction of 9-keto and 15-ketoprostaglandins in rats during pseudopregnancy and pregnancy. The activity of ovarian CR decreased from the onset of pseudopregnancy and pregnancy, reaching 20-30% of the Day 1 value by Day 12 of pseudopregnancy and 50-60% of the Day 1 value by Day 14 of pregnancy. In the case of pregnant rats, the enzyme activity maintained a minimal level between Day 14 of pregnancy and Day 22 of parturition. An acute increase of the enzyme activity was found on the morning after parturition. The CR content in the ovary maintained a constant level from Day 1 to Day 12 of pseudopregnancy and to Day 18 of pregnancy. In pregnant rats, there was a gradual decrease after 18 days and then a surge during parturition. CR was primarily localized in interstitial gland cells and in theca interna cells but was not found in corpora lutea cells in the ovary during the estrous cycle. Additional immunostaining was also observed in corpora lutea cells during pseudopregnancy and pregnancy. The changes in ovarian CR activity, i.e. the rapid decrease with progressing pseudopregnancy and pregnancy, correlated with the increase in progesterone and the decrease in LH. These results indicate that rat ovarian CR may be regulated via the hypothalamo-pituitary-ovarian axis and may also be involved in luteal function.     

Enhanced muscarinic M1 receptor gene expression in the corpus striatum of streptozotocin-induced diabetic rats

Acetylcholine (ACh), the first neurotransmitter to be identified, regulate the activities of central and peripheral functions through interactions with muscarinic receptors. Changes in muscarinic acetylcholine receptor (mAChR) have been implicated in the pathophysiology of many major diseases of the central nervous system (CNS). Previous reports from our laboratory on streptozotocin (STZ) induced diabetic rats showed down regulation of muscarinic M1 receptors in the brainstem, hypothalamus, cerebral cortex and pancreatic islets. In this study, we have investigated the changes of acetylcholine esterase (AChE) enzyme activity, total muscarinic and muscarinic M1 receptor binding and gene expression in the corpus striatum of STZ – diabetic rats and the insulin treated diabetic rats. The striatum, a neuronal nucleus intimately involved in motor behaviour, is one of the brain regions with the highest acetylcholine content. ACh has complex and clinically important actions in the striatum that are mediated predominantly by muscarinic receptors. We observed that insulin treatment brought back the decreased maximal velocity (V_max) of acetylcholine esterase in the corpus striatum during diabetes to near control state. In diabetic rats there was a decrease in maximal number (B_max) and affinity (K_d) of total muscarinic receptors whereas muscarinic M1 receptors were increased with decrease in affinity in diabetic rats. We observed that, in all cases, the binding parameters were reversed to near control by the treatment of diabetic rats with insulin. Real-time PCR experiment confirmed the increase in muscarinic M1 receptor gene expression and a similar reversal with insulin treatment. These results suggest the diabetes-induced changes of the cholinergic activity in the corpus striatum and the regulatory role of insulin on binding parameters and gene expression of total and muscarinic M1 receptors.     

Differential regulation of apoptosis in the corpus luteum of pregnancy and newly formed corpus luteum after parturition in rats

Apoptosis contributes to luteal regression in many species. In the postpartum rat, there are two different types of corpora lutea (CL) in the ovary: CL of pregnancy (CLP) and newly formed CL (NCL). To investigate the regulation of apoptosis in the two different types of CL during luteal regression, apoptosis and caspase-3 activity were examined in the CL obtained on Days 7, 15, and 21 of pregnancy and Days 0, 1, 3, 5, 7, and 9 postpartum. Furthermore, the effect of lactation on apoptosis in the CL was examined in two groups of postpartum rats: lactating rats that nurse more than 10 pups, and nonlactating rats that nurse no pups. Apoptotic cells were detected after Day 21 of pregnancy. In the CLP, remarkable increases in the number of apoptotic cells on Days 5 and 9 postpartum were observed in nonlactating rats (P < 0.01), but not in lactating rats. Changes in caspase-3 activity in the CLP were not consistent with those in number of apoptotic cells. In the NCL, an increase in apoptosis was found only on Day 5 postpartum in nonlactating rats (P < 0.01), but not in lactating rats. Changes in caspase-3 activity in the NCL were consistent with those in number of apoptotic cells. In conclusion, apoptosis is, at least in part, involved in luteal regression after parturition, and lactation appears to inhibit apoptosis. This study also suggests the presence of a caspase-3-independent mechanism for apoptosis in CLP regression in the rat.     

Insulin receptors in developing rat liver: Acquisition of down regulation in the immediate postnatal period

Alterations in the high and low affinity insulin receptor concentrations in developing rat liver were investigated. The number of high affinity receptors in partially purified plasma membranes from fetal rats increased from Days 19 through 22 of gestation, with no further increase in binding during the postnatal period. Fetuses of diabetic rats had approximately three times as many high affinity insulin receptors as age-matched fetuses of normal rats; however, by 1 day after birth the receptor number decreased to the normal level. Neither the number of low affinity receptors nor the affinity of insulin binding to high or low affinity receptors changed during development or between offspring of normal and diabetic rats. These changes in the number of high affinity hepatic insulin receptors from prenatal animals did not correlate with the concentration of plasma insulin. When suckling pups were rendered diabetic the changes in the number of high affinity insulin receptors correlated with alterations in plasma insulin concentrations. The number of high affinity sites/microgram DNA in hepatocytes from Day 18 fetal rats was not altered when cells were cultured for 48 h in medium containing 0, 250, or 5000 μU/ml of added insulin. When cultured hepatocytes derived from 1-day-old and adult rats were maintained in medium with added insulin concentrations of 250 or 5000 μU/ml the number of high affinity receptors/microgram DNA decreased as compared to the number of high affinity receptors in hepatocytes cultured in medium with no added insulin. This decrease in receptor number was accompanied by an increase in the affinity of insulin binding to its high affinity receptors. The data show that (i) only the high affinity insulin receptor number increases in rat liver during the prenatal period, (ii) fetuses of diabetic rats show a greater increase in high affinity receptors than do fetuses of normal animals, and (iii) the phenomenon of down regulation for high affinity insulin receptors is not observed in fetal rat liver, but is acquired in the immediate postnatal period.     

Hormonal induction and regulation of lactose synthetase in mouse mammary gland

The augmentation of lactose synthetase activity during late pregnancy and lactation was measured by using both a tissue-culture assay and a cell-free assay. The results indicated at least a 100-fold augmentation in specific activity between late pregnancy and lactation. The cell-free assay indicated that the activities of both subunits of this enzyme had increased to 20-30% of the value during lactation by the last day of pregnancy. The tissue-culture assay, however, showed activities only 3-4% of the maximum at the time of parturition. This suggests that not all the enzyme present in the tissue before lactation commenced was active. Since at all stages of pregnancy and lactation the B subunit, alpha-lactalbumin (which is also a milk protein), was rate-limiting, it is suggested that the rate of lactose synthesis may be linked to the rate of milk-protein synthesis. Both subunits of lactose synthetase could be induced in tissue culture by the hormones insulin+hydrocortisone+prolactin. Of the three hormones, prolactin appeared to be the ;trigger' that induced the synthesis of these proteins if the tissue had been stimulated previously by insulin+hydrocortisone.     

Metabolism of sheep adipose tissue during pregnancy and lactation. Adaptation and regulation.

1. Changes in the mean volume, the rate of fatty acid and acylglycerol glycerol synthesis, the activity of lipoprotein lipase and the numbers and affinities of insulin receptors of subcutaneous adipocytes are reported for sheep at different stages of pregnancy and lactation. In addition, the serum concentrations of insulin, progesterone, prolactin, choriomammotropin, somatotropin, glucose, acetate, L-lactate, glycerol and unesterified fatty acids are reported for these sheep. 2. A switch from lipid accumulation to net lipid mobilization accompanied by a decline in the capacity for lipid synthesis, occurred at the onset of the last third of pregnancy. Net lipid mobilization continued during lactation. 3. The changes that occurred in the serum concentrations of the various hormones listed above are discussed in relation to their possible roles in the modulation of adipose tissue metabolism in sheep during pregnancy and lactation. The observations are compared with those from previous studies on the hormonal control of adipose tissue metabolism in the rat during pregnancy and lactation.     

Active involvement of PKC for insulin-mediated rates of muscle protein synthesis in Zucker rats

A recent report from our group demonstrated that insulin facilitates muscle protein synthesis in obese Zucker rats. The purpose of this study was to determine whether PKC, a probable modulator of insulin signal transduction and/or mRNA translation, has a role in this insulin-mediated anabolic response. In the first portion of the study, gastrocnemius muscles of lean and obese Zucker rats (n = 5-7 for each phenotype) were bilaterally perfused with or without insulin to assess cytosolic and membrane PKC activity. Limbs perfused with insulin demonstrated greater PKC activity in both lean and obese Zucker rats (P < 0.05) compared with no insulin, but overall activity was greater in obese animals (by approximately 27% compared with lean, P < 0.05). To determine whether PKC plays a role in muscle protein synthesis, hindlimbs (n = 6-8 for each phenotype) were bilaterally perfused with or without insulin and/or GF-109203X (GF; a PKC inhibitor). The presence of GF did not influence the rates of insulin-mediated protein synthesis in gastrocnemius muscle of lean Zucker rats. However, when obese rats were perfused with GF (P < 0.05), the effect of insulin on elevating rates of protein synthesis was not observed. We also used phorbol 12-myristate 13-acetate (TPA, a PKC activator; n = 5-7 for each phenotype) with and without insulin to determine the effect of PKC activation on muscle protein synthesis. TPA alone did not elevate muscle protein synthesis in lean or obese rats. However, TPA plus insulin resulted in elevated rates of protein synthesis in both phenotypes that were similar to rates of insulin alone of obese rats. These results suggest that PKC is a modulator and is necessary, but not sufficient, for insulin-mediated protein anabolic responses in skeletal muscle.     

Studies on the ontogenesis and the regulatory mechanisms of placental lactogen and insulin binding sites (receptor) in rat liver

The ontogenesis of specific binding of 125I-hPL and 125I-insulin was determined in rat liver cell membranes (10(5) X g pellets), and the regulatory mechanisms of these binding sites were also examined. There were striking differences in the mode of ontogenesis between binding sites of hPL and insulin in rats. HPL binding sites were very few in liver cell membranes from fetal and immature rats. They began to increase after puberty, and markedly increased in late pregnancy. On the other hand, insulin binding sites, which decreased in late pregnancy, were dominant in fetal liver and placenta. Consequently, the lipolytic and glycogenolytic activities of hPL in maternal liver were accentuated, whereas the effects of insulin on maternal liver were suppressed. In contrast, in fetal liver and placenta only the anabolic effects of insulin seemed conspicuous. According to the results of experiments on in vivo administration of estradiol-17 beta, progesterone, hydrocortisone or hPL to intact or hypox-rats, and the measurement of serum rat chorionic mammotropin (rCM), rPRL, estradiol-17 beta, and insulin during pregnancy in rats, the increase in hepatic hPL binding sites observed in late pregnancy might be, at least in part, due to rCM secreted from placenta, and the decrease in insulin binding sites due to the increase in serum insulin itself in rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adipose tissue metabolism during early pregnancy in the rat: temporal relationships of changes in the metabolic activity, number of insulin receptors, and serum hormone concentrations

The temporal relationships between changes in rates of fatty acid and acylglycerol glycerol synthesis; the activity of lipoprotein lipase of parametrial adipocytes and their capacity to bind insulin; and the serum concentrations of insulin, progesterone, prolactin, and total lactogenic activity have been examined in rats during the first 15 days of pregnancy. The rate of fatty acid and acylglycerol synthesis showed a transient increase at Days 9 and 12 of pregnancy, whereas there was no change in the activity of lipoprotein lipase activity except for a fall between Days 12 and 15 of pregnancy. The capacity of adipocytes to bind insulin was increased by Day 6 of pregnancy and remained elevated until at least Day 15; no changes in the affinity for insulin were observed. Serum progesterone, insulin, and total lactogenic activity were elevated by Days 3, 9, and 12 of pregnancy, respectively. The results show that progesterone but not placental lactogen could be responsible for the rise in the insulin-binding capacity of rat adipocytes during pregnancy, whereas the fall in lipogenic capacity at about Day 12 of pregnancy coincides with the rise in serum placental lactogen.     

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.     

Changes of sex hormone-binding globulin/SHBG expression in the hypothalamo-hypophyseal system of rats during pregnancy, parturition and lactation.

Sex hormone-binding globulin (SHBG) is expressed in hypothalamic magnocellular neurons. High co-localization rates of SHBG with oxytocin have been observed in the hypothalamus, indicating that SHBG plays a role in pregnancy, parturition and lactation. Further studies have shown that hypothalamic SHBG expression is malleable to changing steroid conditions. In this study, we have examined SHBG levels in the supraoptic and paraventricular hypothalamic nuclei and in the posterior pituitary lobe of late pregnant, parturient and early lactating rats by IN SITU hybridization, immunocytochemistry, and ELISA. Immunocytochemical and biochemical analysis showed that the SHBG levels increased during late pregnancy in hypothalamic nuclei. During parturition, SHBG levels fell in the magnocellular nuclei but increased in the posterior pituitary lobe. SHBG levels increase again during lactation. At day six of lactation, there was no significant difference in SHBG levels compared to normal cycling female rats, which served as control in this study. IN SITU hybridization showed increased SHBG mRNA signal during late pregnancy. The highest SHBG expression was observed during parturition. Our data indicate that hypothalamic SHBG expression changes during pregnancy, parturition and lactation, parallel to ovarian steroid and co-localized OT levels. This may in part be linked to known steroid actions on synthesis and secretion of magnocellular hypothalamic peptide hormones, important for the control of parturition and lactation.     

Changes in activity and intra-acinar distribution of glucose-6-phosphate dehydrogenase and malic enzyme during pregnancy in rat liver

Using techniques of microdissection and microassay as well as qualitative histochemistry the activity and intra-acinar distribution of G6PDH and ME were studied on selected days of pregnancy in the rat. Both enzymes show distinct fluctuations during the course of pregnancy in keeping with changes in hepatic lipogenesis. Marked increases in activity are seen as early as the 4th day, while highest values are attained on day 20, with a predominant perivenous induction. On day 22, just before parturition a sharp decrease of both enzyme activities with a flattening of the periportal/perivenous gradient was detected. G6PDH shows proportionally considerably larger increases and more distinct changes in zonation. The perivenous fluctuations in G6PDH activity of late gestation are supposed to be caused primarily by insulin. Although estrogen is known to induce both enzymes, the temporal changes in enzyme activity in pregnancy cannot be related to the action of estrogen alone. The changes in enzyme activity, however, correspond well to those of progesterone, and although no direct action of progesterone on these enzymes has yet been proposed, further work on its effects on enzyme activity and distribution is indicated.     

Changes in the activities of enzymes of the biosynthetic pathway of the nicotinamide nucleotides in rat mammary gland during the lactation cycle

1. The activities of NMN pyrophosphorylase, NMN adenylyltransferase and NAD kinase in the mammary glands of rats at different stages of pregnancy, lactation and involution were measured. 2. NMN pyrophosphorylase has a low activity early in pregnancy, but its activity increases at parturition and in early lactation to reach a maximum at the tenth day of lactation, after which it remains constant until it declines abruptly in involution. 3. NMN adenylyltransferase is already quite active by the tenth day of pregnancy and its activity does not rise further in the second half of gestation. After a sharp rise in activity at parturition, the activity of the enzyme declines slowly throughout the period of lactation and, more sharply, in involution. 4. NAD kinase has a low activity for most of pregnancy, but its activity rises at parturition to a value at 2 days of lactation that is maintained until the tenth day. Between the tenth and fifteenth days of lactation the activity almost doubles, but falls sharply in mammary involution. 5. The relation of the activities of these enzymes to the rates of synthesis of NAD and NADP is discussed.     

Regulation of myometrial beta 2-adrenergic receptors by progesterone and estradiol-17 beta in late pregnant rats

Rat myometrium exhibited a marked decrease in the concentration of beta 2-adrenergic receptors immediately before parturition, i.e., in the last 6 h of pregnancy. This phenomenon continued until the withdrawal of myometrial progesterone (-94% from Day 18 of pregnancy to term) and coincided with the sharp increase (+200%) of the myometrial concentration of estradiol. A linear positive correlation was found (r2 = 0.645) between the concentration of beta 2-adrenergic receptors and the log ratio of myometrial concentration of progesterone/myometrial concentration of estradiol (P/E2), suggesting a modulation of beta 2-adrenergic receptors by steroids. In rats with estrogen-dominated uteri (intact of ovariectomized late pregnant rats injected with estradiol), there was no change either in concentration or affinity of beta 2-adrenergic receptors relative to untreated control pregnant rats. In contrast, rats with progesterone-dominated uteri (intact or ovariectomized late pregnant rats treated with progesterone or ovariectomized rats) have an increased number of beta 2-adrenergic receptors, with a decreased affinity of these receptors compared to untreated control pregnant rats or to estrogen-treated rats. These results suggest that progesterone regulates the number of beta 2-adrenergic receptors in myometrium of late pregnant rats. The mechanisms by which progesterone exerts this regulation remains to be elucidated.     

Fat accumulation in the rat during early pregnancy is modulated by enhanced insulin responsiveness

Insulin sensitivity has been implicated in the variation of fat accumulation in early gestation by as-yet-unknown mechanisms. In the present study, we analyzed the insulin sensitivity of lipolysis and lipogenesis in lumbar adipocytes from rats at 0, 7, 14, and 20 days of gestation. In adipocytes of 7-day pregnant rats, we found a twofold decrease in both beta-agonist (isoproterenol and BRL-37344)-stimulated lipolysis and beta3-adrenoceptor protein but not in lipolysis initiated by forskolin or isobutylmethylxanthine, suggesting a modification of the lipolytic pathway at the receptor level. Whereas adipocytes from 7-day pregnant rats showed a twofold increase in fatty acid synthesis from glucose, those from 20-day pregnant animals displayed a decreased lipogenic activity. Insulin responsiveness of the lipolytic and lipogenic pathways was analyzed by dose-response experiments, giving evidence for the involvement of improved insulin responsiveness in the enhanced lipogenic and reduced lipolytic activities of adipocytes in early pregnancy. In contrast, insulin resistance is responsible for lower antilipolytic and lipogenic actions of insulin in late pregnant animals. In conclusion, the present study shows that enhanced adipose tissue insulin responsiveness during early pregnancy contributes to maternal fat accumulation, whereas decreased insulin responsiveness during late gestation modulates fat breakdown.     

Aberrant regulation of the metabolism of the insulin receptor in Swarm rat chondrosarcoma chondrocytes.

Treatment of Swarm rat chondrosarcoma chondrocytes for 3 days in media containing either non-recombinant pig or recombinant human insulin (1 micrograms/ml) increased the rate of proteoglycan synthesis approximately 6-fold compared with cells cultured in the absence of insulin. The concentrations of human and pig insulin that stimulated the cells to double their rate of proteoglycan synthesis were approximately 1 ng/ml and approximately 2 ng/ml respectively. Because physiological concentrations of insulin do not influence proteoglycan synthesis in non-transformed chondrocytes, the findings indicated a possible abnormality in the insulin-dependent regulation of the insulin receptor in these tumour cells. Like most cells, chondrosarcoma chondrocytes down-regulated their insulin receptors when incubated with insulin for 30 min. However, the number of plasma-membrane and intracellular insulin receptors did not decrease when the chondrocytes were exposed to insulin chronically for 4 days. Chondrocytes were cultured in media containing 2H-, 13C- and 15N-labelled amino acids, and the heavy-isotope density-shift method was used to investigate both the rate of degradation and the rate of synthesis of the insulin receptor. Although the rate of synthesis of the receptor was slightly faster in the insulin-treated cultures, as assessed by a slightly faster rate of appearance of the 'heavy' receptor, the rate of degradation of the receptor was slower in the insulin-treated cultures. The half-lives for the 'light' receptors were approx. 18 h and 10 h for chondrocytes cultured in insulin-containing and insulin-free media respectively. These studies in vitro indicate that the apparent up-regulation of insulin receptors that occurs in this transformed cell upon long-term exposure to insulin is primarily the result of a decreased rate of receptor degradation.     

Plasma prolactin,progesterone and oestradiol-17β concentrations around parturition in the pig

Plasma concentrations of prolactin, progesterone and oestradiol-17β were measured by radioimmunoassay in samples taken from 2–15 days before until 1–4 days after spontaneous parturition in four sows and in one sow around prostaglandin F2α-induced parturition.Between Days ?15 and ?2 (Day 0 = parturition), prolactin concentrations in daily samples fluctuated somewhat, but exceeded 10 ng/ml only exceptionally. Plasma progesterone levels gradually declined or remained high until about 2 days before parturition. A final decrease of the progesterone concentrations coincided with distinct increases of the prolactin levels during the last 40 h of pregnancy.Maximal prolactin concentrations were measured before the onset of delivery of the piglets. Oestradiol-17β reached peak values around delivery. Prostaglandin F2α injection caused an immediate sharp increase of the prolactin concentration which lasted for about 6 h. During this period progesterone and oestradiol-17β concentrations did not change. A second elevation of prolactin levels was measured when progesterone finally decreased.Changes of prolactin concentrations found in this study were compared with those found in other domestic animals at the same reproductive stage.