Re-examination of the putative roles of insulin and prolactin in the regulation of lipid deposition and lipogenesis in vivo in mammary gland and white and brown adipose tissue of lactating rats and litter-removed rats.

1. The effects of various treatments to alter either plasma prolactin (bromocryptine administration or removal of litter) or the metabolic activity of the mammary gland (unilateral or complete teat sealing) on the disposal of oral [14C]lipid between 14CO2 production and [14C]lipid accumulation in tissues of lactating rats were studied. In addition, the rates of lipogenesis in vivo were measured in mammary gland, brown and white adipose tissue and liver. 2. Bromocryptine administration lowered plasma prolactin, but did not alter [14C]lipid accumulation in mammary gland or in white and brown adipose tissue. 3. In contrast, complete sealing of teats results in no change in plasma prolactin, but a 90% decrease in [14C]lipid accumulation in mammary gland and a 4-fold increase in white and brown adipose tissue. The rate of lipogenesis in mammary gland was decreased by 95%, but there was no change in the rate in white and brown adipose tissue. Unilateral sealing of teats resulted in a decrease in [14C]lipid accumulation in white adipose tissue. 4. Removal of the litter for 24 h (low prolactin) produced a similar pattern to complete teat sealing, except that there was a 6-fold increase in lipogenesis in white adipose tissue. Re-suckling for 5 h increased plasma prolactin, but did not alter the response seen in litter-removed lactating rats. 5. Changes in lipoprotein lipase activity and in plasma insulin paralleled the reciprocal changes in [14C]lipid accumulation in white and brown adipose tissue and in mammary gland. 6. It is concluded that the plasma insulin is more important than prolactin in regulating lipid deposition in adipose tissue during lactation, and that any effects of prolactin must be indirect.     

Effects of glucose-containing peritoneal-dialysis solutions on rates of lipogenesis in vivo in the liver, brown and white adipose tissue of chronic uraemic rats.

Chronic uraemic rats had decreased food intake, and this was accompanied by decreased weight of the epididymal fat-pads and interscapular brown adipose tissue. Normal rats whose food intake was restricted to an amount similar to that of the uraemic rats showed similar decreases in weight of the adipose-tissue depots. In addition, the food-restricted rats had decreased liver weight compared with normal or uraemic rats. The basal rate of lipogenesis was decreased in liver and epididymal fat-pads of food-restricted and uraemic rats and in interscapular brown adipose tissue of uraemic rats. Administration of a low-glucose-containing (1.36%) peritoneal-dialysis solution slightly increased lipogenesis in liver of uraemic rats, but had no significant effect in epididymal fat-pads. For brown fat, the rate of lipogenesis was increased in normal, food-restricted and uraemic groups, but the values for the last group were 4-5-fold lower than for the food-restricted or control groups. A high-glucose-containing (3.86%) peritoneal-dialysis solution gave similar rates of lipogenesis in liver, epididymal fat-pads and brown fat of all three groups, but for brown fat moderately uraemic rats showed a considerably lower rate of lipogenesis than did mildly uraemic rats. The basal plasma insulin concentration was lower in the food-restricted (50%) and uraemic (70%) groups than in the control group. The low-glucose peritoneal-dialysis solution increased plasma insulin to control values in the food-restricted rats, but had no significant effect on plasma insulin in the uraemic rats, despite a significant increase in blood glucose in this group. It is concluded that there is an impairment of the lipogenic response to intraperitoneal glucose loads in interscapular brown adipose tissue of uraemic rats, and that this is not due to the accompanying decrease in food intake. The hypoinsulinaemia may be an important factor. The possible relevance of this finding to the obesity observed in some uraemic patients treated by peritoneal dialysis with glucose-containing solutions is discussed.     

Effect of premature weaning on amino acid uptake by the mammary gland of lactating rats.

1. Arteriovenous differences of amino acids across the lactating mammary gland were measured in normal rats and weaned for 4, 5 and 24h. 2. Uptake of amino acids by mammary glands of rats weaned for 5h or more was significantly lower than that of controls. This was not reversed by injection of prolactin. 3. By using 'unilaterally weaned' rats we showed that milk accumulation plays an important role in amino acid uptake by mammary gland. 4. gamma-Glutamyltransferase activity was significantly lower in 'weaned' glands than in 'normal' glands. This provides further support for the hypothesis of the function of the gamma-glutamyl cycle in the mammary gland in vivo.     

The role of prolactin and progesterone in the regulation of lipogenesis in maternal and foetal rat liver in vivo and in isolated hepatocytes during the last day of gestation.

The administration of progesterone on day 21 of gestation increases the rates of lipogenesis in the liver in vivo and in hepatocytes isolated from rats on day 22 of pregnancy. Bromocriptine administration increases the rates of hepatic lipogenesis in vivo, but has no effect on lipid synthesis in hepatocytes under the same treatment conditions. Concurrently, the administration of progesterone or bromocriptine on day 21 to the mother increases the rates of lipogenesis in the foetal liver in vivo on day 22. The rates of lipid synthesis in foetal isolated hepatocytes are increased by progesterone administration, but remain unchanged by bromocriptine.     

Differences in the regulation of adipose tissue and liver lipogenesis by carbohydrates in humans

We assessed the contributions of human liver and adipose tissue de novo lipogenesis (DNL) to triacylglycerol (TAG) synthesis. Volunteers were fed a high-energy, high-carbohydrate diet (HC, n = 5) or a normocaloric diet (NC, n = 10). NC subjects remained in the fasting state (Study 1, n = 5) or received oral glucose (Study 2, n = 5) throughout the test (12 h). HC subjects remained in the fasting state (Study 3). They ingested deuterated water and [U-13C]acetate to trace lipogenesis. Adipose tissue fatty-acid (FA) synthase (FAS), acetyl-CoA carboxylase 1 (ACC1), and SREBP-1c mRNA were measured. Plasma TAG-FA was labeled by 13C and deuterium showing active liver lipogenesis, which was stimulated (P < 0.05) by oral glucose and HC diet. Adipose tissue TAG had no detectable 13C enrichment in any test, showing no significant incorporation of TAG-FA provided by liver lipogenesis, but were labeled by deuterium in all tests, showing active DNL in situ; however, rough quantitative estimates showed that adipose DNL was minimal (<1 g), and poorly stimulated by oral glucose or HC diet. mRNA levels were not increased by the HC diet. Adipose DNL is active in humans, but contributes little to TAG stores and is less responsive than liver DNL to stimulation by carbohydrates.     

Important sites of lipogenesis in the mouse other than liver and white adipose tissue

In a cell-free system prepared by osmotic lysis of protoplasts of Cephalosporium acremonium, isopenicillin N is converted into penicillin N. The epimerase activity of the system is labile.     

In vivo lipogenesis and enzyme levels in adipose and liver tissues from pair-fed genetically obese and lean rats

Genetically obese Zucker rats pair-fed to lean controls were similar in body weight and food intake, however, epididymal fat pads were considerably larger than lean controls. $\text{In}$$\text{vivo}$ incorporation of acetate-¹⁴C into adipose tissue lipid was not significantly different, however, $\text{in}$$\text{vivo}$ liver lipogenesis was elevated in the obese rat. Characterization of enzyme profiles in both liver and adipose tissues revealed that enzymes normally associated with lipogenesis were elevated in liver tissue from obese rats. Malic enzyme and citrate cleavage enzyme were both depressed in adipose tissue of obese animals. From these data, it appears that the liver may be prominently involved in the development of excessive blood lipid and enlarged fat cells in the Zucker obese rat.     

Insulin-like effects of fluoroacetate on lipolysis and lipogenesis in adipose tissue.

Hormone-stimulated lipolysis in adipose tissue was inhibited by fluoroacetate and there was a concomitant decrease in both the basal and hormone-stimulated cyclic AMP levels. Adenylate cyclase (EC 4.6.1.1) activity in membrane preparations was inhibited by fluoroacetate. There was no influence of fluoroacetate on the low Km cyclic AMP phosphodiesterase (EC 3.1.4.17) activity. The rate of glucose conversion to fatty acids was increased when adipose tissue was incubated in the presence of fluoroacetate. The outputs of pyruvate and lactate into the incubation medium were decreased at this time, suggesting decreased tissue pyruvate levels and a site of activation of lipogenesis distal to pyruvate formation. Pyruvate dehydrogenase (EC 1.2.4.1) activity was increased twofold in adipose tissue incubated in the presence of fluoroacetate. This was attributed to a fluoroacetate-induced inhibition of pyruvate dehydrogenase kinase, the enzyme responsible for inactivating the pyruvate dehydrogenase complex. Glucose transport was increased to a small but significant degree by fluoroacetate. In addition, both the tissue content of citrate and its release into the incubation medium were increased, suggesting that fluoroacetate resulted in an inhibition of aconitase (EC 4.2.1.3). The tissue ATP content was unchanged. Because the antilipolytic and lipogenic effects of fluoroacetate parallel those of insulin, they may share a common mechanism.     

Protein malnutrition of the lactating female rat: effect on maternal behaviour and spontaneous weaning of young rats

Female rats were kept on a diet with a low content of animal protein (5.5%) beginning from the 2nd day after delivery. Beginning from the 10th day after delivery their body weight was persistently lower than that of control females. Also the body weight of their young was lower than that of control animals throughout the experiment, with significant differences appearing on the 17th day of life. Spontaneous termination of maternal milk intake occurred in malnourished young 10 days later than in the controls. Retention of 85Sr in malnourished females was in keeping with their milk production. Irrespective of the differences in body weight and the quality of solid food, the malnourished young began consuming solid food at the same age and in the same relative amounts as the controls. Thus in malnourished young the period of gradual weaning was prolonged by more than to 3 weeks in contrast to 2 weeks in the controls. This is probably due to the slower extinction of the oral sucking mechanism resulting from changes in the maturation of the CNS. No changes were found in maternal behaviour (nest building and efficiency of retrieving) in malnourished females during the suckling period. However, infanticide appeared in some females during the weaning period.     

Diurnal variations in food intake and in lipogenesis in mammary gland and liver of lactating rats.

Despite the hyperphagia, the food intake of the lactating rat showed marked diurnal changes which paralleled those of virgin rats. The major difference was that lactating rats consumed a higher proportion (35%) of their diet during the light period than did virgin rats (14%). The peak rate of lipogenesis in the lactating mammary gland occurred around midnight, and this decreased by 67% to reach a nadir around mid-afternoon; this corresponded with the period of lowest food intake. The diurnal variations in hepatic lipogenesis in lactating rats were much less marked. The changes in hepatic glycogen over 24 h suggest that it acts to supply carbon for lipogenesis during the period of decreased food intake. The activation state of acetyl-CoA carboxylase in mammary gland altered during 24 h, but the changes did not always correlate with alterations in the rate of lipogenesis. The changes in plasma insulin concentration tended to parallel the food intake in the lactating rats, but they did not appear to be sufficient to explain the large alterations in lipogenic rate in the mammary gland.     

The effects of meal-feeding and the diurnal cycle on lipogenesis in brown adipose tissue of rats

A significant diurnal variation in the rates of lipogenesisin vivo in brown adipose tissue occurred in both virgin and lactating rats. On a meal-feeding regime of either a chow, high-sucrose, or high-lipid diet, there was a very large increase in BAT lipogenesis following the meal. The rates observed after the sucrose meal are the highest so far reported. There was no significant difference in BAT lipogenesis between lactating and virgin rats, contrary to previous reports by others. The pattern of stimulation of BAT lipogenesis by these feeding regimes was different from that for white adipose tissue and liver and was not correlated with plasma insulin levels.     

Developmental changes of lipogenic enzyme activities and lipogenesis in brown adipose tissue and liver of the rat.

1. Brown adipose tissue (BAT) and liver lipogenesis in vivo estimated by using 3H2O as tracer was very low and did not change significantly between 10 and 20 days after birth. Lipogenesis increased dramatically in both tissues by weaning at 20 days, peaking between 25 and 30 days of age. Since that time the rate of fatty acid synthesis in BAT decreased gradually to reach adult level after 2 months, whereas in the liver there was a sharp decrease of lipogenesis. 2. The activities of fatty acid synthase, citrate cleavage enzyme, malic enzyme and glucose 6-phosphate dehydrogenase essentially followed a similar course of developmental changes as lipogenesis. 3. In contrast to the enzymes listed above NADP-linked isocitrate dehydrogenase remained unaltered over the period studied, whereas lactate and malate dehydrogenases exhibited very high activity at 10 days after birth and from then decreased to reach adult level at the age of about 20 days. 4. The data obtained indicate that no substantial differences could be detected in the developmental pattern of lipogenesis and lipogenic enzyme activities between BAT and liver up to 30 days of age but after this time these processes were not co-ordinated in both tissues. Beyond this time the BAT was characterized by a much higher rate of lipogenesis than the liver. 5. The results are discussed in terms of the nutrient changes and the relationship between thermogenesis and lipogenesis in BAT.     

Increased lipoprotein lipase content in the adipose tissue of suckling and weaning obese Zucker rats.

The aim of this study was to determine whether the increase in lipoprotein lipase activity displayed by the adipose tissue of obese (fa/fa) rats as compared with that of lean (Fa/fa) rats could be ascribed to a change in the content or in the catalytic properties of the enzyme. The question was addressed in rats of two ages: in 7-day-old suckling and in 30-day-old post-weaning pups. Inguinal fat-pads were removed surgically (7 days of age) or after killing (30 days of age), and acetone-extract powders were prepared. The relative quantity of enzyme was assessed by immunotitration using an antiserum raised in goat against purified lipoprotein lipase from rat adipose tissue. The results indicate that increases in enzyme activity in obese animals were strictly paralleled by increases in the amount of enzyme in suckling as well as in post-weaning pups. Moreover, the apparent Km values of lipoprotein lipase for its substrate triacylglycerol were identical in the two genotypes. In conclusion, the genotype-mediated increase in lipoprotein lipase activity in adipose tissue of obese Zucker rats was fully accounted for by an increase in the content of the enzyme. In addition, this work documents the mechanism of the increase in lipoprotein lipase activity during weaning, which is mediated mainly through changes in the adipose-tissue enzyme content.     

Effects of inhibition of protein synthesis by cycloheximide on lipogenesis in mammary gland and liver of lactating rats.

1. Administration of cycloheximide (an inhibitor of protein synthesis) to lactating rats raised the concentrations of amino acids, and in particular, the branched-chain amino acids (valine, leucine and isoleucine) in blood, liver and mammary gland. 2. Inhibition of protein synthesis increased the incorporation in vivo of L-[U-14C]leucine into lipids of mammary gland and liver. 3. Cycloheximide treatment caused no immediate change in the overall rate of lipogenesis in vivo (measured with 3H2O) in mammary gland but increased the rate in liver 3-fold; this latter effect also occurred in livers of virgin rats. 4. The increased rate of hepatic lipogenesis was not accompanied by significant changes in the plasma insulin concentration or the activity of acetyl-CoA carboxylase. 5. Although cycloheximide decreased the entry of total triacylglycerol into the circulation it did not alter the rate of secretion of newly synthesized saponifiable lipid. 6. Cycloheximide slightly stimulated lipogenesis from endogenous substrates in isolated hepatocytes, but this effect was abolished when lactate was the exogenous substrate. 7. Administration of cycloheximide to virgin rats decreased liver glycogen and increased the hepatic content of glucose 6-phosphate, pyruvate and lactate. 8. It is concluded that (a) there is no short-term link between the rate of protein synthesis and lipogenesis in the lactating mammary gland and (b) the increased rate of hepatic lipogenesis in cycloheximide-treated rats is mainly due to stimulation of glycogenolysis, glycolytic flux and consequent increased availability of pyruvate.     

Possible chondroregulatory role of prolactin on the tibial growth plate of lactating rats

Besides calcium accretion in the cortical envelope, a marked increase in the length of long bone was observed in pregnant and lactating rats, and thus the growth plate change was anticipated. Since several bone changes, such as massive trabecular bone resorption in late lactation, were found to be prolactin (PRL)-dependent, PRL may also be responsible for the maternal bone elongation. Herein, we investigated the growth plate change and possible chondroregulatory roles of PRL in the tibiae of rats at mid-pregnancy until 15 days postweaning. We found that the tibial length of lactating rats was increased and was inversely correlated with the total growth plate height, as well as the heights of proliferating zone (PZ) and hypertrophic zone (HZ), but not the resting zone (RZ). Chondrocytes in all zones expressed PRL receptors as visualized by immunohistochemistry, suggesting that the growth plate cartilage was a target of PRL action. Further investigations in lactating rats treated with an inhibitor of pituitary PRL release, bromocriptine, with or without PRL supplement, revealed the PRL-induced decreases in total growth plate height and HZ height from early to late lactation. However, decreases in RZ and PZ heights were observed only in late and mid-lactation, respectively. Thus, this was the first report on the chondroregulatory action of PRL on the growth plate of long bone in lactating rats. The results provided better understanding of the maternal bone adaptation during lactation.     

Effect of somatotropin on adipose tissue net glucose-stimulated lipogenesis in young goats.

Net glucose-stimulated lipogenesis (NGSL: the rate of lipogenesis in the presence of glucose minus the rate of lipogenesis in the absence of glucose) in omental adipose tissue explants from young castrated male goats was evaluated in control animals (n = 3; placebo-treated) and in animals treated with the sustained release of recombinant bovine somatotropin (n = 4; bST; 100 mg at 7-day intervals in a 147 days lasting experiment). The rate of fatty acid synthesis was determined in acute incubations in both freshly prepared and chronically cultured explants. Adipose explants remained metabolically active and retained their ability to respond to hormones when maintained in a tissue culture medium. NGSL in explants cultured for 24 h in the presence of insulin alone or bST alone, was non-significantly increased (more in the controls) and decreased (more in bST-treated animals), respectively. However, cortisol alone decreased (P<0.05) NGSL in explants from both control and bST-treated animals. In tissues from bST-treated animals, cortisol acted synergistically with insulin to produce a higher rate of NGSL than that observed in cultures with insulin alone. bST inhibited insulin plus cortisol-stimulated lipogenesis significantly (P<0.05) in explants from bST-treated animals but non-significantly in control animals. The rates of NGSL were decreased (P<0.05) by catecholamines in explants from both control and bST-treated animals. Norepinephrine (NE) and isoprenaline (ISO) were equally effective in the controls, whereas isoprenaline was more effective than norepinephrine in bST-treated animals.     

Evidence for a role of insulin in the regulation of lipogenesis in lactating rat mammary gland. Measurements of lipogenesis in vivo and plasma hormone concentrations in response to starvation and refeeding.

Fatty acid synthesis in the mammary gland of lactating rats in vivo was 5-fold higher than in the liver. Starvation decreased fatty acid synthesis in the gland 50-fold, whereas refeeding for 2h completely reversed this change. The plasma insulin concentration decreased 2-fold in starvation and was restored to the fed-rat value on refeeding. Glucagon and prolactin concentrations did not always change in parallel with lipogenesis, suggesting that insulin may be a regulator of this process in the gland.     

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.