The utilization of ketone bodies by the interscapular brown adipose tissue of the rat

The activities of 3-oxo acid-CoA transferase (EC 2.8.3.5, 13-15 micromol/min per g) and acetoacetyl-CoA thiolase (EC 2.3.1.9, 18-21 micromol/min per g) in interscapular brown adipose tissue of the rat are comparable to the activities reported for heart and kidney. The incorporation of D-3-hydroxy[3-14C]butyrate into lipid in vivo was about 30-fold higher in interscapular brown adipose tissue than in white adipose tissue of virgin rats. In lactating rats, the mammary gland was the major site of ketone body incorporation into lipid and incorporation of D-3-hydroxy-[3-14C]butyrate into lipid in brown adipose tissue was lower than in virgin rats. After an oral load of medium chain triacylglycerol, which inhibits lipogenesis in lactating mammary gland, the incorporation of ketone bodies into lipid was decreased in mammary gland but increased in brown adipose tissue. The rate of oxidation of D-3-hydroxy[3-14C]butyrate by brown adipose tissue slices in vitro was higher than the rate of incorporation into lipid.     

Acute effects of endotoxin (lipopolysaccharide) on tissue lipid metabolism in the lactating rat. The role of delivery of intestinal glucose

The aim of this study was to compare the effects of endotoxin on lipid metabolism and, in particular, lipogenesis in virgin and lactating rats. Intraperitoneal administration of bacterial endotoxin (lipopolysaccharide, LPS; 3 mg/kg body wt.) to fed virgin rats caused a 4-fold increase in lipogenic rate in liverin vivo. The stimulatory effect was not seen when glucose (6 mmol) was administered either orally or intraperitoneally to increase the basal rate. In contrast, the rate of lipogenesis in interscapular brown adipose tissue was inhibited, after LPS, and this was relieved by intraperitoneal glucose. In the lactating rat there were no significant changes in hepatic lipogenesis after the administration of endotoxin. However, LPS decreased the lipogenic rate in mammary gland of lactating rats and intraperitoneal glucose administration, but not oral, was able to restore the rate. In both virgin and lactating rats, LPS decreased glucose removal from the intestina tract. In lactating rats, LPS induced a rise in blood concentrations of lactate, and plasma triacylglycerols and non-esterified fatty acids, similar to those in endotoxin-treated virgin rats. The administration of LPS did not decrease the accumulation of radioactivity in lipid in either liver or in mammary gland after injection of³H-oleate. In contrast, LPS decreased the accumulation of radioactivity in mammary gland after injection of²H-chylomicrons and increased it in liver and plasma. These changes were accompanied by a decrease in mammary gland activity of lipoprotein lipase. Intraperitoneal glucose partially reversed these changes in chylomicron disposition. It is concluded that the inhibitory effect of LPS on mammary gland lipogenesis and uptake of exogenous lipid is primarily due to sensitivity of this tissue to the rate of delivery of glucose from the intestine.     

Tissue-specific effects of starvation and refeeding on the disposal of oral [1-14C]triolein in the rat during lactation and on removal of litter.

1. The effects of starvation and refeeding on the disposal of oral [14C]triolein between 14CO2 production and 14C-lipid accumulation in tissues of virgin rats, lactating rats and lactating rats with pups removed were studied. 2. Starvation (24 h) increased 14CO2 production in lactating rats and lactating rats with pups removed to values found in virgin rats. This increase was accompanied by decreases in 14C-lipid accumulation in mammary gland and pups of lactating rats and in white and brown adipose tissue of lactating rats with pups removed. 3. Short-term (2 h) refeeding ad libitum decreased 14CO2 production in lactating rats and lactating rats with pups removed, and restored the 14C-lipid accumulation in mammary glands plus pups and in white and brown adipose tissue respectively 4. Insulin deficiency induced with mannoheptulose inhibited the restoration of 14C-lipid accumulation in white adipose tissue on refeeding of lactating rats with pups removed, but did not prevent the restoration of 14C-lipid accumulation in mammary gland. 5. Changes in the activity of lipoprotein lipase in mammary gland and white adipose tissue paralleled the changes in 14C-lipid accumulation in these tissues. 6. It is concluded that 14C-lipid accumulation in mammary gland may not be affected by changes in plasma insulin concentration and that it is less sensitive to starvation than is lipogenesis or lactose synthesis. This has the advantage that the milk lipid content can still be maintained from hepatic very-low-density lipoprotein for a period after withdrawal of food. The major determinant of the disposal of oral 14C-triolein appears to be the total tissue activity of lipoprotein lipase. When this is high in mammary gland (fed lactating rats) or white adipose tissue (fed lactating rats with pups removed), less triacylglycerol is available for the muscle mass and consequently less is oxidized.     

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.     

Tissue-specific effects of rapid tumour growth on lipid metabolism in the rat during lactation and on litter removal.

1. The effect of tumour burden on lipid metabolism was examined in virgin, lactating and litter-removed rats. 2. No differences in food intake or plasma insulin concentrations were observed between control animals and those bearing the Walker-256 carcinoma (3-5% of body wt.) in any group studied. 3. In virgin tumour-bearing animals, there was a significant increase in liver mass, blood glucose and lactate, and plasma triacylglycerol; the rate of oxidation of oral [14C]lipid to 14CO2 was diminished, and parametrial white adipose tissue accumulated less [14C]lipid compared with pair-fed controls. 4. These findings were accompanied by increased accumulation of lipid in plasma and decreased white-adipose-tissue lipoprotein lipase activity. 5. In lactating animals, tumour burden had little effect on the accompanying hyperphagia or on pup weight gain; tissue lipogenesis was unaffected, as was tissue [14C]lipid accumulation, plasma [triacylglycerol] and white-adipose-tissue and mammary-gland lipoprotein lipase activity. 6. On removal (24 h) of the litter, the presence of the tumour resulted in decreased rates of lipogenesis in the carcass, liver and white and brown adipose tissue, decreased [14C]lipid accumulation in white adipose tissue, but increased accumulation in plasma and liver, increased plasma [triacylglycerol] and decreased lipoprotein lipase activity in white adipose tissue. 7. The rate of triacylglycerol/fatty acid substrate cycling was significantly decreased in white adipose tissue of virgin and litter-removed rats bearing the tumour, but not in lactating animals. 8. These results demonstrate no functional impairment of lactation, despite the presence of tumour, and the relative resistance of the lactating mammary gland to the disturbance of lipid metabolism that occurs in white adipose tissue of non-lactating rats with tumour burden.     

Tumour necrosis factor alpha (cachectin) mimics some of the effects of tumour growth on the disposal of a [14C]lipid load in virgin, lactating and litter-removed rats.

Tumour necrosis factor alpha (cachectin) was administered to virgin, lactating and litter-removed rats, and subsequent disposal of an oral [1-14C]triolein (glycerol tri[1-14C]oleate) load examined. Absorption of the lipid and 14CO2 production were significantly depressed in all three groups. [14C]Lipid accumulation was decreased in carcass, liver and adipose tissue (brown and white) of virgin and litter-removed rats and the mammary gland of lactating rats. The plasma triacylglycerol concentration was increased in all three groups, and lipoprotein lipase activity was decreased in the white adipose tissue of virgin and litter-removed animals and in the mammary gland of lactating animals. Some, but not all, of these effects mimic tumour burden in the same physiological states [Evans & Williamson (1988) Biochem. J. 252, 65-72].     

Lactogenic and somatogenic binding sites in intact and detergent-solubilized membrane preparations of female rat liver.

1. Lactation results in decreased glucose and acetate utilization and increased lactate output by sheep adipose tissue. 2. The ability of insulin to stimulate acetate uptake was lost in adipose tissue from lactating sheep, whereas both the response and the sensitivity (ED50) for insulin for stimulation of glucose conversion into products other than lactate were decreased. These impairments were partly restored by prolonged incubation of adipose tissue for 48 h. 3. The ability of insulin to stimulate lactate output was not altered by lactation. 4. Dexamethasone inhibited glucose uptake, lactate output and glycerol output in adipose tissue from both non-lactating and lactating sheep, with an ED50 of about 1 nM. Dexamethasone inhibited acetate uptake by adipose tissue from non-lactating sheep, but this effect was not observed with adipose tissue from lactating sheep. 5. Dexamethasone inhibited the stimulation of glucose uptake at all concentrations of insulin used; the effect varied with insulin concentration and resulted in an accentuation of the insulin dose-response curve. The insulin dose-response curve in the presence of dexamethasone was muted during lactation. 6. The overall effect of these adaptations is to ensure that glucose and acetate utilization by adipose tissue after an insulin surge is diminished during lactation.     

Integration of lipid metabolism in the mammary gland and adipose tissue by prolactin during lactation

Prolactin deficiency, induced by bromocryptine treatment, brought about reciprocal changes in the ability of adipocytes and acini isolated from lactating rats to synthesize lipids. The capacity to synthesize fatty acids and phospholipids decreased in the mammary gland and increased in adipocytes by bromocryptine treatment. In the mammary gland, the maximum potential activity of the pentose shunt as well as the specific activities of the pathway dehydrogenases were significantly reduced by bromocryptine treatment. Simultaneously, adipose tissue increased its lipogenic capacity but neither the maximum potential of the shunt nor the specific activities of the pentose phosphate shunt dehydrogenases were significantly changed with respect to the control lactating rats. Thus, a differential regulatory mechanism(s) of the pentose phosphate shunt activity appears to operate in these two tissues. Adipocytes from lactating rats showed a poor responsiveness to insulin in terms of lipid synthesis from glucose. In contrast, in adipocytes from bromocryptine treated rats insulin was able to increase lipid synthesis (105%). Sheep prolactin administration in vivo partially reversed the effects of bromocryptine. These data suggest that prolactin mediates adipocytes resistance to insulin during lactation. Phospholipid synthesis, as occurred in fatty acid synthesis, is increased in adipose tissue and decreased in mammary gland by bromocryptine treatment. However, -adrenergic stimulation increases phosphatidylinositol turnover to about the same percentages in both mammary gland acini and adipocytes from lactating rats independently of bromocryptine treatment.     

Influence of starvation/refeeding transition on lipogenesis and NADPH producing systems in adipose tissue, mammary gland and liver at mid-lactation

Glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and malic enzyme are enzymes involved in NADPH synthesis. Their specific activities and glucose utilization by isolated cell systems have been measured in adipose tissue and mammary gland from mid-lactating rats during starvation/refeeding transition. Starvation for 24 h produced a 75-90% decrease in the specific activities of these NADPH producing systems in mammary gland. Acinis isolated from the gland of starved rats had a lower production of CO2, fatty acids and triacylglycerols from (1-14C)glucose and (6-14C)-glucose than did gland from control rats. The activities of these enzymes in adipose tissue were very low and did not undergo any measurable alteration with starvation. The ability of adipocytes from well fed lactating rats to synthesize fatty acids from (1-14C)glucose was completely blocked. However, starvation is accompanied by a marked decrease in glucose incorporation into triacylglycerols. All the variations observed "in vivo" and "in vitro" in mammary gland returned almost to normal values by refeeding the starved lactating rats.     

Interactions of glucose, acetoacetate and insulin in mammary-gland slices of lactating rats.

1. Utilization of 5mM-glucose by slices of lactating mammary gland was decreased 33% on addition of acetoacetate (2mM) to the incubation medium. This inhibition was accompanied by increases in the intracellular concentrations of citrate and glucose 6-phosphate. 2. In the presence of acetoacetates the accumulation of pyruvate in the medium approximately doubled. 3. Insulin completely reversed the inhibitory effect of acetoacetates on glucose utilization, without altering the amount of acetoacetate removed or pyruvate formed. 4. Similar results were obtained with mammary-gland slices from diabetic rats, except that insulin did not completely reverse the effects of acetoacetates. 5. Acetoacetate inhibited the formation of 14CO2 from [1-14C]pyruvate; this effect was not overcome by insulin. 6. Insulin increased the proportion of [3-14C]acetoacetate that was converted into lipid and decreased that oxidized to CO2.7. The physiological significance of these findings is discussed.     

Regulation of fatty acid synthesis in lactating rat mammary gland in the fed to starved transition: asynchronous control of pyruvate dehydrogenase, phosphofructokinase and acetyl-CoA carboxylase.

1. Withdrawal of food from lactating rats produced a rapid and dramatic decrease in the uptake of glucose by the mammary gland and an inhibition of the rate of fatty acid synthesis that could not be explained alone by decreased substrate supply to the tissue. 2. Within the first 6 hr starvation, fatty acid synthesis and pyruvate dehydrogenase activity were inhibited by 87 and 80%, respectively, but acetyl-CoA carboxylase activity did not change significantly. 3. Between 6 and 24 hr starvation, total and expressed activities of acetyl-CoA carboxylase decreased by 62 and 55%, respectively. 4. The ratio of fructose-6-phosphate/fructose-1,6-bisphosphate concentration in mammary tissue increased 9-fold during the first 6 hr starvation, indicating an inhibition of 6-phosphofructo-1-kinase. However, the major inhibition of this enzyme occurred between 6 and 24 hr starvation when this metabolite ratio increased a further 160-fold in parallel with increased tissue citrate concentration. 5. The increase in citrate concentration between 6 and 24 hr starvation correlated with acetyl-CoA carboxylase inactivation and ketone body accumulation in the mammary gland. 6. This study confirms the asynchronous control of three important regulatory steps in the pathway of glucose utilization and fatty acid synthesis in the lactating rat mammary gland.     

The regulation of lipogenesis in vivo in the lactating mammary gland of the rat during the starved-refed transition. Studies wtih acarbose, a glucosidase inhibitor.

Depression of carbohydrate digestion by oral administration of acarbose, a glucosidase inhibitor, led to a 75% inhibition of the re-activation of lipogenesis in vivo in the mammary gland of 18 h-starved lactating rats refed with 5 g of chow diet. Rates of [1-14C]glucose incorporation in vitro into lipid and CO2 in mammary-gland acini isolated from refed animals were elevated compared with acini from starved rats, but acarbose treatment completely prevented this stimulation. Gastric intubation of glucose led to a large stimulation of lipogenesis in the mammary gland of starved lactating rats, similar to that induced by refeeding with chow diet; this was dependent on the amount of glucose given and the time elapsed between glucose administration and injection of 3H2O for the measurement of lipogenesis. The switch-on of lipogenesis in the mammary gland of starved lactating rats, by refeeding or by intubation of glucose, was associated with a decrease in the ratio of [glucose 6-phosphate]/[fructose 1,6-bisphosphate] in the gland, indicative of an increase in phosphofructokinase activity. A time-course study revealed that the ratio decreased rapidly over the first 30 min of chow refeeding, after which a large surge in lipogenesis was seen. Acarbose, given 25 min after the onset of refeeding, led to a stepwise increase in the ratio, in parallel with the observed decrease in lipogenic activity. It is concluded that the control of lipogenesis in the mammary gland is closely linked to the availability of dietary carbohydrate. An important site of regulation of lipogenesis in the gland appears to be at the level of phosphofructokinase. A possible role of insulin in the regulation of phosphofructokinase activity, and the acute modulation of insulin-sensitivity in the gland during the starved-refed transition, are discussed.     

Lipogenesis in interscapular brown adipose tissue of virgin, pregnant and lactating rats. The effects of intragastric feeding.

Lipogenesis in brown adipose tissue of virgin rats increased 8--10-fold after intragastric feeding with glucose or medium-chain triacylglycerol, and this increase was prevented by short-term insulin deficiency. Brown adipose tissue increased in weight during pregnancy, regressed during lactation and hypertrophied again on weaning; the rate of lipogenesis paralleled these changes. Glucose did not increase brown-adipose-tissue lipogenesis at mid-lactation.     

Regulation of lactating-rat mammary-gland lipogenesis by insulin and glucagon in vivo. The role and site of action of insulin in the transition to the starved state.

Starvation for 6h and 24h caused an 80% and 95% decrease in the rate of mammary-gland lipogenesis respectively in conscious lactating rats. 2. Plasma insulin concentrations decreased and circulating ketone-body concentrations increased with the length of starvation. 3. The inhibition of lipogenesis after 24h starvation was accompanied by increased concentrations of glucose, glucose 6-phosphate and citrate in the mammary gland. Qualitatively similar changes were observed after 6h starvation. 4. Infusion of insulin at physiological concentrations caused a 100% increase in the rate of lipogenesis in fed animals and partially reversed the inhibition of lipogenesis caused by starvation. 5. Infusion of insulin tended to reverse the changes seen in intracellular metabolite concentrations. 4. Infusion of glucagon into fed rats caused no change in the rates of lipogenesis in mammary gland, liver or white adipose tissue. 7. It is concluded that (a) insulin acts physiologically to regulate lipogenesis in the mammary gland, (b) hexokinase and phosphofructokinase are important regulatory enzymes in the short-term control of lipogenesis in the mammary gland, which are under the influence of insulin, and (c) the unresponsiveness of mammary-gland lipogenesis in vivo to infusions of glucagon is consistent with an adaptive mechanism which diverts substrate towards the lactating mammary gland and away from other tissues.     

Changes in the properties of cytosolic acetyl-CoA carboxylase studied in cold-clamped liver samples from fed, starved and starved-refed rats.

The mechanism responsible for the insulin resistance described in vivo in brown adipose tissue (BAT) of lactating rats was investigated. The effect of insulin on glucose metabolism was studied on isolated brown adipocytes of non-lactating and lactating rats. Insulin stimulation of total glucose metabolism is 50% less in brown adipocytes from lactating than from non-lactating rats. This reflects a decreased effect of insulin on glucose oxidation and lipogenesis. However, the effect of noradrenaline (8 microM) on glucose metabolism was preserved in brown adipocytes from lactating rats as compared with non-lactating rats. The number of insulin receptors is similar in BAT of lactating and non-lactating rats. The insulin-receptor tyrosine kinase activity is not altered during lactation, for receptor autophosphorylation as well as tyrosine kinase activity towards the synthetic peptide poly(Glu4-Tyr1). The defect in the action of insulin is thus localized at a post-receptor level. The insulin stimulation of pyruvate dehydrogenase activity during euglycaemic/hyperinsulinaemic clamps is 2-fold lower in BAT from lactating than from non-lactating rats. However, the percentage of active form of pyruvate dehydrogenase is similar in non-lactating and lactating rats (8.6% versus 8.9% in the basal state, and 37.0% versus 32.3% during the clamp). A decrease in the amount of pyruvate dehydrogenase is likely to be involved in the insulin resistance described in BAT during lactation.     

Physiological significance of altered insulin metabolism in the conscious rat during lactation.

Uptake of radioactively labelled insulin by the mammary gland of the rat increased 12-fold in lactation compared with non-lactating controls. This uptake was decreased by the presence of unlabelled insulin, indicating that it occurred via insulin receptors. The plasma half-life of insulin is decreased in lactation from 9.4 min to 4.8 min, and the metabolic clearance rate for insulin increased from 7.26 to 13.03 ml/kg body wt. per min. The basal insulin and glucose concentrations in the plasma were decreased in lactation. Infusion of insulin at a dose which led to a small physiological rise in plasma insulin concentration increased lipogenic rates in the mammary gland by 100% without causing marked hypoglycaemia. It is concluded that the lactating mammary gland is a highly insulin-sensitive tissue and that the lower plasma insulin during lactation occurs primarily as a result of this sensitivity increasing extraction of glucose by the gland and thus producing a decrease in the plasma glucose concentration. It is suggested that a secondary result of the fall in plasma insulin concentration is the preferential direction of substrates (glucose and non-esterified fatty acids) towards the lactating mammary gland and away from adipose tissue and the liver.     

Monosaccharide transport in the mammary gland of the intact lactating rat.

The Michaelis-Menten equation for the utilization of competing substrates was applied to the uptake of 2-deoxy[3H]glucose into the mammary gland of anaesthetized lactating rats. Intracellular water was calculated from total tissue water and sucrose space. Fed rats had a mean transport capacity of 2.2 mumol/min per g of tissue, giving an actual glucose transport in vivo of 1.1 mumol/min per g. Transport decreased by 90% on overnight starvation and returned to normal by 2 h of re-feeding. Similar changes were observed in the 1 min or 5 min transport of circulating 3-O-methylglucose. Transport of 3-O-methylglucose in starved rats was restored towards normal by insulin. In fed rats it increased between parturition and day 12 of lactation. The findings support the proposal that transport is a rate-limiting factor in the mammary utilization of carbohydrate.     

Comparative effects of insulin and proinsulin in vitro on pathways of glucose utilization and lipid synthesis in the lactating rat mammary gland

The effects of insulin and proinsulin have been measured on the rates of glucose oxidation via the pentose phosphate pathway and incorporation into lipid in slices of lactating rat mammary gland. Half-maximal stimulation of glucose oxidation was observed with 1-3 x 10(-8)M insulin while 1 x 10(-7)M proinsulin was required to achieve half-maximal stimulation. A similar, approximately 10-fold, difference in potency was observed in relation to lipid synthesis. The present results appear to indicate that the maximum stimulation of either glucose oxidation via the pentose phosphate pathway or lipid synthesis by proinsulin did not reach the same level as that found for insulin.     

Alterations in the activities of rat tissue hexose monophosphate dehydrogenases in response to premature weaning and dietary restriction at mid-lactation

Summary The activities of the hexose monophosphate dehydrogenases increased in adipose tissue, remained unchanged in liver and decreased in mammary gland following the weaning of rats at mid-lactation (day 14). When dietary intake was restricted at mid-lactation, the activities of the hexose monophosphate dehydrogenases increased in adipose tissue, decreased in liver, but were unaltered in mammary gland. Premature weaning on day 14 postpartum resulted in maternal increases in both plasma insulin and glucose, which peaked at day 16. The plasma insulin levels decreased from day 14 to day 18 postpartum in the normal lactating rat, and a similar trend was observed for animals on a restricted dietary intake. Daily food consumption in the lactating rat decreased from 50 g to 20 g after premature weaning. The live weight of pups raised on dams given a restricted food intake from day 14 had decreased by day 17 postpartum, whereas an increase in daily live weight gain was recorded for the litters from the lactating controls. The results demonstrate that the activities of the hexose monophosphate dehydrogenases are regulated differentially between tissues of the lactating rat.     

Vanadate activates pentose phosphate pathway and glycolysis, and raises fructose 2,6-bisphosphate concentration in slices of lactating rat mammary gland.

In mammary gland slices from lactating rats, vanadate increased the rate of glucose oxidation via the pentose phosphate pathway by 36% and raised the glucose flux via glycolysis by 47%. Furthermore, vanadate increased the fructose 2,6-bisphosphate (Fru-2,6-P2) level by 33%. The effect of vanadate on glucose oxidation was compared to the effect of insulin. The present data indicate that 0.5mM vanadate has an effect on glucose utilization similar to that of insulin but does not reach the same level.