Insulin resistance of hind-limb tissues in vivo in lactating sheep.

1. The effects of varying the plasma insulin concentration by infusion while maintaining euglycaemia by infusion of glucose on nutrient arterio-venous differences across the hind-limb and mammary gland in lactating and non-lactating sheep were investigated. 2. Insulin infusion increased the glucose arterio-venous difference across the hind-limb; this effect of insulin was decreased by lactation, suggesting that lactation induces insulin resistance in skeletal muscle. 3. Lactation increased but insulin infusion decreased the plasma concentrations of acetate, beta-hydroxybutyrate and non-esterified fatty acids. 4. Insulin infusion decreased the arterio-venous differences of acetate and hydroxybutyrate across the hind-limb; this effect of insulin is probably indirect, resulting from the decrease in plasma concentrations of these metabolites. 5. Infusion of insulin had no effect on the glucose arterio-venous difference across the mammary gland, but did decrease the oxygen arterio-venous difference. 6. The results suggest that lactation results in insulin resistance in skeletal muscle, at least with respect to glucose utilization; this should facilitate the preferential utilization of glucose by the mammary gland.     

Insulin affects glucose uptake by muscle and mammary tissues of lactating ewes

Effects of insulin on exchanges of glucose across skeletal muscle and mammary tissue were measured in short-term studies in lactating ewes. Insulin secretion was suppressed by a primed/continuous infusion of somatostatin, then insulin was administered by continuous intravenous infusion of doses that were increased, in a step-wise manner, from 0 to 2 U h-1. Plasma glucose was maintained essentially constant by frequent monitoring and intravenous administration of exogenous glucose. Somatostatin suppressed but did not completely inhibit insulin secretion as shown by maintenance of plasma concentration of C-peptide. As plasma insulin was increased, while arterial glucose was maintained stable, uptake of glucose by skeletal muscle increased and glucose uptake by the mammary gland decreased. These observations confirm the role of insulin in regulating glucose uptake by skeletal muscle and raise the possibility that insulin also regulates glucose uptake by the mammary gland.     

Changes in bovine mammary insulin binding during pregnancy and lactation

1. Binding of insulin to microsomes from mammary glands of pregnant and lactating dairy cows was characterized. 2. Binding affinities of the insulin receptor did not change from pregnancy to lactation. 3. Maximal specific binding occurred in microsomes from cows in mid-pregnancy and declined in microsomes from cows in late pregnancy. 4. Insulin binding continued to decrease from early to mid-lactation and increased during late lactation. 5. Results indicate that decreased sensitivity in mammary tissue from lactating dairy cows is at least in part a result of a reduction in insulin receptor number. 6. Results demonstrate further physiological differences between the ruminant and non-ruminant mammary gland.     

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.     

Polymyxin B diminishes blood flow to brown adipose tissue and lactating mammary gland in the rat. Possible mechanism of its action to decrease the stimulation of lipogenesis on refeeding.

Polymyxin B, a cyclic decapeptide antibiotic, increased blood glucose and lactate, and inhibited the stimulation of lipogenesis in interscapular brown adipose tissue and lactating mammary gland of starved-refed virgin and lactating rats respectively. Lipogenesis was not inhibited in white adipose tissue or liver. The antibiotic increased the haematocrit. The relative blood flow to brown adipose tissue and lactating mammary gland was decreased by polymyxin B, and this was accompanied by a decrease in tissue ATP content. In vitro polymyxin B did not affect glucose utilization or conversion into lipid, nor the stimulation by insulin of these processes in brown-adipose-tissue slices. Treatment of rats in vivo with polymyxin B resulted in decreased utilization of glucose in vitro in brown-adipose-tissue slices. Similarly, acini from mammary glands of polymyxin B-treated lactating rats had decreased rates of conversion of [1-14C]glucose to lipid. It is concluded that the effects of polymyxin B may be brought about by decreases in tissue blood flow. The possibility that these effects are secondary to inhibition of glucose utilization cannot be ruled out.     

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.     

Arteriovenous glucose differences across the mammary gland of the fed, starved, and re-fed lactating rat.

Arteriovenous glucose difference across the mammary gland of the lactating rat was used as an 'instantaneous' monitor of mammary glucose uptake. Plasma [glucose] and arteriovenous glucose difference varied according to whether Halothane, diethyl ether or sodium pentobarbitone anaesthesia was used. In pentobarbitone-treated rats a 60% glucose extraction in the fed state decreased to 5% after 18 h starvation, and recovered to 40% and 59% after 15 min and 60 min re-feeding respectively. The increase and decrease in plasma [fatty acids] and the depletion and restoration of hepatic glycogen mostly followed similar time courses. Re-feeding was accompanied by a brief surge of plasma [insulin]. Starved lactating rats showed a markedly greater capacity than age-matched virgin rats in the oral and intraperitoneal glucose tolerance tests. Mammary glucose uptake in the starved rat was significantly restored by oral or intraperitoneal glucose or by insulin, but not by acetoacetate or by heparin-induced elevation of plasma [fatty acids]. The role of insulin and of possible changes in mammary sensitivity to insulin in the return of mammary glucose uptake on re-feeding is discussed.     

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.     

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.     

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.     

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.     

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.     

Phosphoribosyl pyrophosphate and phosphoribosyl pyrophosphate synthetase in rat mammary gland. Changes in the lactation cycle and effects of diabetes, insulin and phenazine methosulphate.

Changes in the tissue content of phosphoribosyl pyrophosphate (PPRibP), glucose 6-phosphate, ribose 5-phosphate (Rib5P), RNA and DNA, of the activity of PPRibP synthetase (EC 2.7.6.1) and the conversion of [1-14C]- and [6-14C]-glucose into 14CO2 were measured at mid-lactation in the normal and diabetic rat and in pregnancy, lactation and mammary involution in the normal rat. The PPRibP, glucose 6-phosphate and Rib5P contents increase during pregnancy and early lactation to reach a plateau value at mid-lactation, before falling sharply during weaning. The PPRibP content, PPRibP synthetase activity and flux of glucose through the oxidative pentose phosphate pathway (PPP) all change in parallel during the lactation cycle. Similarly, after 3 and 5 days duration of streptozotocin-induced diabetes, ending on day 10 of lactation, there were parallel declines in PPRibP content, PPRibP synthetase and PPP activity. The effect of streptozotocin was prevented by pretreatment with nicotinamide and partially reversed by insulin administration. Addition of insulin to lactating rat mammary-gland slices incubated in vitro significantly raised the PPRibP content (+47%) and the activity of the PPP (+40%); phenazine methosulphate, which gives a 2-fold increase in PPP activity, raised the PPRibP content of lactating mammary gland slices by approx. 3-fold. It is concluded that Rib5P, generated in the oxidative segment of the PPP, is an important determinant of PPRibP synthesis in the lactating rat mammary gland and that insulin plays a central role in the regulation of the bioavailability of this precursor of nucleotide and nucleic acid synthesis.     

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.     

Glucose transporter expression in rat mammary gland.

The expression of different glucose transporter isoforms was measured during the development and differentiation of the rat mammary gland. Before conception, when the mammary gland is mainly composed of adipocytes, Glut 4 and Glut 1 mRNAs and proteins were present. During pregnancy, the expression of Glut 4 decreased progressively, whereas that of Glut 1 increased. In the lactating mammary gland only Glut 1 was present, and was expressed at a high level. The absence of Glut 4 suggests that glucose transport is not regulated by insulin in the lactating rat mammary gland.     

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.     

Rapid inhibition by intragastric triolein of the re-activation of glucose utilization and lipogenesis in the mammary gland during the starved-refed transition in lactating rats. Evidence for a direct effect of oral lipid on mammary tissue.

1. Oral administration of triacylglycerol (triolein) to starved/chow-refed lactating rats suppressed the lipogenic switch-on in the mammary gland in vivo. 2. A time-course study revealed that triolein, administered at 30 min after the onset of refeeding, had no influence on lipogenic rate in the mammary gland between 30 and 60 min, but markedly decreased it between 60 and 90 min. Glucose uptake by the mammary gland (arteriovenous difference) increased by 30 min of refeeding, as did lactate production. Between 30 and 90 min glucose uptake remained high in the control animals, but glucose uptake and net C3-unit uptake were decreased in the triolein-loaded animals by 90 min. 3. Triolein increased [glucose 6-phosphate] in the gland and simultaneously decreased [fructose 1,6-bisphosphate], indicative of a decrease in phosphofructokinase activity. This cross-over occurred at 60 min, i.e. immediately before the inhibition of lipogenesis, and by 90 min had reached 'starved' values. 4. Triolein had no effect on plasma [insulin] nor on whole-blood [glucose], [lactate] or [3-hydroxybutyrate]; a small increase in [acetoacetate] was observed. 5. Infusion of the lipoprotein lipase inhibitor, Triton WR1339, abolished the suppression of mammary-gland lipogenesis by triolein and the increase in the [glucose 6-phosphate]/[fructose 1,6-bisphosphate] ratio, suggesting a direct influence of dietary lipid on mammary-gland glucose utilization and phosphofructokinase activity.     

Effects of serum on mammary epithelial proliferation in vitro during mammary gland development

The proliferative response of mammary gland epithelium from nonpregnant, pregnant, and lactating mice to mammary serum factor and insulin was studied in vitro. Mammary gland epiithelium from nonpregnant and lactating animals has a delayed proliferative response to mammary serum factor and insulin when compared to the response of epithelium from pregnant animals. The results show that as the animals go through pregnancy into lactation the mammary gland epithelium becomes less responsive to mammary serum factor while it retains its responsiveness to insulin. The concentration of mammary serum factor in sera from animals at various physiological stages is constant. Sera from hypophysectomized rats, on the other hand, show a 50% drop in mammary serum factor activity. This loss of activity cannot be reversed by injecting prolactin, 17-beta-estradiol, or growth hormone into the hypophysectomized animals. A hypothesis that the mammary gland is composed of two proliferative epithelial populations is developed, and the possible role of prolactin in stimulating DNA synthesis is discussed.     

Physiological significance of the concentration of human milk glucose

Summary The concentration of glucose in human milk, [Glucose]_milk, was directly related to the volume of milk secreted not only during lactogenesis and weaning but also during full lactation. To investigate the mechanism for this observation we first established that glucose equilibrates across the apical membrane of the mammary alveolar cell, using infusion of stable isotopically labelled glucose into lactating women. Our results indicate that [Glucose]_milk can be used to measure the glucose concentration in the mammary alveolar cell, [Glucose]_cell. We then investigated the regulation of glucose transport into the mammary alveolar cell using glucose clamp methodology in fully lactating and weaning women. Maintenance of high plasma insulin concentrations for four hours under euglycemic conditions had no effect on [Glucose]_milk, demonstrating that insulin does not regulate glucose transport into the mammary gland. On the other hand, maintaining the [Glucose]_plasma at twice the fasting level resulted in a 3-fold increase in the steady state [Glucose]_milk in fully lactating women and a 5-fold increase in [Glucose]_milk in weaning women. Kinetic analysis of the data showed that the V_max for glucose transport into the mammary alveolar cell across the basolateral membrane is regulated by the level of synthetic activity in the mammary alveolar cell.Dedicated to Professor Stuart Patton on the occasion of his 70th birthday.     

Insulin regulation of amino-acid metabolism in the mammary gland of sheep in early lactation and fed fresh forage

Insulin plays an important role in regulating the partitioning of nutrients to the mammary gland, particularly in lactating ruminants fed concentrate-based diets. There is evidence that the nutritional status of the animals might also affect their response to insulin. This is largely untested in early lactating ruminants fed fresh forage. To investigate nutritional effects on insulin response, 12 lactating sheep, housed indoors, were allocated to one of two treatment groups (hyperinsulinaemic euglycaemic clamp (HEC) or control) in a randomised block design and fed perennial ryegrass (Lolium perenne)/white clover (Trifolium repens) pasture. Mammary amino acid (AA) net uptake from plasma and utilisation for milk protein synthesis was measured during the 4th day of the HEC using arterio-venous concentration differences, and 1-13C-leucine was used to estimate whole body and mammary gland leucine kinetics. There was no change in feed intake, milk protein output and mammary blood flow during the HEC (P > 0.1). The HEC decreased (P < 0.1) the arterial concentrations of all essential AA (EAA) except histidine. The mammary net uptake of some EAA (isoleucine, leucine, methionine and phenylalanine) was reduced by the HEC (P < 0.1). Leucine oxidation in the mammary gland was not altered during the HEC (P > 0.1) but mammary protein synthesis was reduced by the HEC (P < 0.05). These results show that sheep mammary gland can adapt to changing AA precursor supply to maintain milk protein production during early lactation, when fed fresh forage. How this occurs remains unclear, and this area deserves further study.