Cellular Energy Metabolism and Regulation

SYMPOSIUM: NONMAMMARY METABOLISM IN SUPPORT OF LACTATION AND GROWTH

Cellular Energy Metabolism and Regulation J. M. KELLY, M. SUMMERS, H. S. PARK, L. P. MILLIGAN, and B. W. YcBRlDE Oeparbnent of Animal and Poultry Science University of Guelph, Guelph, ON, Canada N1G ZWl

&phosphate, ODC = ornithine decarboxylase, PEG = polyethylene glycol, PEP-Pyr-OM = phosphoenolpyruvate-pyruvate-oxaloacetate, R M R = resting metabolic rate, TG = triacylglycerol, T3 = triiodothyronine, T4 = thyroxine.

ABSTRACT

Consistent with the increased demand for nutrients imposed by lactation and growth, those tissues directly involved in the digestion, absorption, and processing of the required additional nutrients show response to these states. During lactation, the rumen, upper intestine, and liver increase in size, and more energy is spent on Na+, K+ transport and on protein turnover. The massive endocrine influences during lactation suggest that the metabolism of other tissues besides these and mammary tissue would be influenced, but evidence is rather sparse. Ion transport and protein metabolism in some muscles may indeed be increased. Although substrate cycles characteristically account for a substantially smaller portion of the energy expenditure in the intact animal than do ion transport and protein turnover, stage of lactation influences some of these cycles, particularly the triacylglycerol fatty acid cycle. The needs for additional quantitative in vivo measurements of metabolic conversions and for mechanistic model description of metabolic events in nonmammary tissues are discussed. (Key words: metabolism, lactation, cellular energy)

INTRODUCTION

The topic of this symposium is something of a milestone because it very clearly indicates that at the metabolic level formation of animal products is a great deal more complex than the occurrence of i n d digestive, absorptive, and synthetic conversions in addition to the metabolic array that accounts for maintenance. Thus, even though production and maintenance have been treated as additive in nutritional practice, metabolic understanding of production and the effective use of nutrients for production require recognition of the en* breadth of a metabolic state that is changed from that of maintenance. This paper emphasizes metabolic events in support of lactation with inclusions of changes in p w t h . The reader should also refer to a recent detailed review of growth metabolism by Lobley (90). DIGESTIVE TRACT MORPHOLOGY

(Key words: Abbreviation key: AcCoA = acetyl coenzyme A, EGF = epidermal growth factor, FBP = fructose l,&bisphosphate, FSR = fractional synthetic rate, F6P = fructose 6-phosphate, GDP = guanosine diphosphate, GIT = gastrointestinal tract, GL = glucose, GTP = guanosine triphosphate, G6P = glucose

Received Septemb 29, 1989. Acccpted Fkbruary 5, 1990. 1991 J Dairy Sci 74678-694

Numerous physiological changes occur during lactation, including hypertrophy of the liver, intestine, and rumen epithelium (46,48) and an increase in voluntary feed intake (51). Investigations carried out with ewes demonstrated that the weight of the nuninal mucosa and its total DNA and RNA content increased after parturition and reached maximum values at 45 d postpartum. Also, the mean length of the ruminal papillae was increased, and a si@cant positive correlation was found between the amount of food consumed during the final week of life with both the weight of the ruminal mucosa and the length of ruminal papillae

678

SYMPOSIUM: NONMAMMARY METABOLISM

(111). Others have reported that a striking feature of lactation in various animals is the increase in food intake and the hypertmphy and hyperplasia of the gastrointestinal mucosa (26, 39, 63, 78, 88, 146). Crean and Rumsey (39) observed that hyperplasia of gastric mucosa during pregnancy and lactation in rats entailed substantial increases in the d a c e area and parietal and peptic cell populations of this tissue. After investigation of the role of gastrin and food intake as mediators of duodenal mucosal growth in the lactating rat, Lichtenberger and Trier (88) found that the incrtaSes in crypt cell proliferation were highly correlated with food intake but not with serum gastrin levels. Their results are consistent with other studies on food intake regulation (26, 51, 89, 146). Furthermore, Takeuchi and Johnson (146) demonstrated that increased serum gastrin levels and gastrin binding capacities of the oxyntic gland mucosa in lactating rats were abolished by blocking increased food intake. They also suggested that the mechanisms of hypertrophic responses of the gastric mucosa in lactating rats are significantly comlated with the increased number of gastrin receplors. Because of the well-known relationship between food intake and prolactin levels (1, 22, 65, 93, 94, 139), prolactin was investigated as a physiological cue involved in the stimulation of food intake in lactating rats (53). Gerardo-Gettens et al. (53) demonstrated that cumulative food intake was greatest at the high hormone dose (20% increase), intermediate at the medium dose (17%). and smallest at the low dose (11%) compared with the control group. Even though food intake and weight increased in prolactintreated rats, prolactin would not be the only stimulus for hyperphagia in lactating animals. Considerable evidence indicates that polyamines play a central role in the regulation of gastrointestinal growth (79). Polyamines in the diet or synthesized in the gastrointestinal tract (GIT) can stimulate mucosal growth from the lumen. Investigating ornithine decarboxylase (ODC),the enzyme catalyzing the decarboxylation of omithine to form putrecine and the ratelimiting enzyme in polyamine biosynthesis, Yang et al. (163) showed that mucosal ODC activity in lactating rats increased with increasing morphological gastrointestinal adaptation. They also suggested that ODC activity plays an

IN SUPPORT OF LACTATTON AND GROWTH

679

important but not well-understood role in mucosal hyperplasia during the stimulation of crypt cell proliferation. Similarly, previous studies demonstrated that levels of ODC and polyamines in rapidly growing cells were higher than those in slowly growing or quiescent cells (145). Indeed, ODC has been intensively studied for its rapid response to various stimuli, including growth factors, hormones, drugs, and tumor promotors, which can induce increased ODC activity in various tissues, including duodenal, jejunal, ileal, and colonic mucosae (42,49,92, 109, 163). In various cell types, Na+ influx and H+ efflux across cell membranes (Na+,H+ exchange) p e d e incmses in ODC activity and initiation of DNA synthesis responding to growth stimuli. UlrichBaker et aL (150). using refeeding induction of mucosal cell hyperplasia, demonstrated that the Na+,H+ antiport is essential for the increased ODC activity in the jejunum and for the stimulation of DNA synthesis in the jejunum and liver. Even though the precise mechanism by which gut growth is regulated during lactation is unknown,the increased proliferation of intestinal epithelial cells could relate to more rapid absorption of nutrients in lactating animals. NUTRIENT ABSORPTION

In addition to mucosal hyperplasia accompanied by increased villus height and crypt cell depth, adaptation also occurs in the absorption of amino acids, glucose, water, and minerals in lactating animals (38, 40, 79, 81, 123, 137). Absorption of nutrients ingested from the intestinal lumen can OCCUT by diffusion across the brush border plasma membrane or by means of facilitated transporters, specific transport systems that are usually characterized as secondary active transport proteins (74). Monosaccharides or amino acids may be ab&s by both transport pathways. The active transport of sugars or amino acids follows a sequence of membrane events (69). Sugars or amino acids may be moved into the cell via a Na+-symport system energized by the electrochemical Na+ gradient at the brush border attached to the luminal plasma membrane. Thus, Na+, K+-ATF’ase at the basolateral membrane is necessary to maintain the electrochemical Na+ gradient across the plasma membrane. The accumulated nutrients in the epithelial cells loamal of Dairy Scicnce Vol. 74. No. 2, 1991

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KELLY

could move to blood via facilitated transport. The proportional contribution of the active mechanism for any nutrient, however, could vary with the concentration of the nutrient in the lumen. In addition to its function along the gut, Na+, K+transport plays an essential role in transporting sugars and amino acids across other animal cell membranes (69). Karasov (80) suggested that nutrient uptake in the GlT could respond to changes in the quantity of food consumed per day, the qualitative profiles of food, and the nutritional or physiological state of the animal. As discussed above, lactation causes significant gastrointestinal hyperphagia and endocrinological shifts, which consequently may trigger intestinal transport changes. Cripps and Williams (40) clearly showed that absolute or per unit length absorption of leucine and glucose were sigruficantly increased in the small intestine of the lactating rat. Recently, Karasov (80) provided evidence that the rate of glucose and proline uptake per centimeter of intestine was increased markedly in lactating mice. The adaptations occurring in the GIT may facilitate the further uptake and absorption to meet the increased energetic and nutrient demands during lactation (81, 82). HEPATIC METABOLISM

In early lactation, a variety of interacting factors modify hepatic function. Liver weight increases by about 40% during lactation (3, 16, 27, 104, 112), and a shift in enzyme activity related to the metabolism of carbohydrates and lipids occurs with this hypertrophy (161). In ruminants, glucose utilization by nonsplanchnic, nonmammary tissues decreases mssibly reflecting a reduced rate of lipogenesis in adipose tissue; (153)], and the glucose production by the liver increases (140). Several studies have demonstrated increased activities of those enzymes involved in hepatic gluconeogenesis in response to lactation in sheep and cattle (103, 138, 154), whereas the activities of gluconeogenic enzymes in lactating rats do not change (glucose-6-phosphatase; G6Pase) or show a decrease [phosphoenolpyruvate carboxykinase; (161)l. Furthermore, in the ruminant studies the activity of several hepatic glycolytic enzymes remained constant during lactation, whereas the activity of glucokinase (glycolytic) J o d of Dairy Science Vol. 74, No. 2, 1991

-- .increased in lactating rats (161). The increase in liver weight during lactation is markedly correlated to mobilization of body reserves in early lactation cows (159). PROTEIN TURNOVER

Tissues undergoing increases in DNA synthesis and cell division during lactation and growth have an associated increase in protein turnover. Most intracellular proteins are in a constant state of flux, accretion being the consequence of protein synthesis exceeding degradation. These two components of protein turnover, first described by Schoenheimer et al. (134), contribute substantially to the energetics of the cell (72, 142). Lactation is an alteration from the maintenance state and includes alterations in protein turnover. Intracellular protein degradation is achieved by at least two distinct means: the ubiquitin ATP-dependent cytosolic proteolysis and lysosomal protein breakdown. To our knowledge, no direct estimates have been made of body protein degradation in the lactating animal. Estimates have been calculated from differences in tissue accretion and fractional rates of protein synthesis. Energetics

The energetic cost of protein synthesis assumes that the rate of activation of amino acids is equivalent to the synthesis rate of peptide bonds (128). Peptide bond synthesis per se usually is considered to entail at least three major energetically demanding steps per peptide bond formed (117). Formation of the aminoacyl-tRNA occurs with the consequent hydrolysis of ATP to AMP,which is equivdent to use of two high energy phosphate bonds. Initiation, at the cost of one high energy phosphate bond, involves the positioning of the amino acid at the correct site on the ribosome. Elongation, the extension of the amino acid chain, also entails the use of another high energy phosphate bond (117). In addition, an energetic cost (.33 ATP) is associated with the transport of the amino acid across the cell membrane (107). Other associated energy demanding costs during protein synthesis include the formation of the initiation complex (1 17) and the turnover of RNA and DNA (120,

SYMPOSIUM: NONMAMMARY METABOLISM IN SUPPORT OF LACTATION AND GROWTH

136), although neither the rates nor stoichiome-

try of nucleic acid turnover has been elucidated (142).

A distinct requirement of energy for protein degradation has been demonstrated in the ubiquitin system (68). Rapport et al. (126) indicated that the ubiquitin-stimulated degradation of protein in reticulocytes had an energetic cost of 1 mol of ATP per peptide bond hydrolyzed. This value was also utilized by Gill et al. (54) in their model of energy expenditures in the growing lamb. Physiological State

Different physiological states can have dramatic effects on the energetics of protein tumover, the stoichiometry of ATP per peptide bond synthesized or degraded is assumed to be unchanged (54). In disease states such as diabetes, a low rate of muscle protein synthesis is exhibited (52), but after administration of insulin protein synthesis is restored to n o d levels (118). Once insulin is removed from these diabetic rats, there is a corresponding loss of muscle protein, depressed muscle protein synthesis, and a rise in protein degradation (119). Protein turnover is influenced by the health of the animal. Hasselgren et al. (66) indicated a 90% decline in a-aminoisobutyric acid uptake into the soleus and cardiac muscles and a 45% decline in uptake into the diaphragm resulting from sepsis in rats. This condition is also associated with an increased muscle proteolysis and release of amino acids to the circulation (35), providing further evidence that protein metabe lism is altered by metabolic state. Cold exposure, which is associated with increased thyroid status and elevated heat production (86, 102. 160), increases protein turnover in mice (164) and chicks (2) and increases the rate of skeletal muscle protein breakdown in cattle (147). The increases in protein metabolism contribute to the increased 0 2 consumption that occurs during cold exposure. Adaptation of protein metabolism during lactation has received considerably less attention in the literature than that during growth and development, even though a marked nutritional stress may occur (21) and nutrient re quirements increase substantially (155). During peak lactation, in contrast to the virgin state, the mammary gland is the main site of amino

681

acid metabolism exhibiting a sixfold increase in [l%]leucine incorporation into mammary gland protein (156); little information is available on amino acid and protein metabolism in tissues other than the mammary gland during lactation (162). Because of the absolute high energetic cost of milk synthesis (55), coupled with the large increase in intake (36) and alterations in whole body and nonmammary energy metabolism in support of milk synthesis (110), real need exists to generate information that quantitatively compares the metabolic modifications occurring in nonmammary tissues in the lactating with the nonlactating state, especially for ruminants. Adaptations of tissues to lactation are related to the service functions of the tissue. Increasing the level of intake in rats raises the visceral organ mass of RNA and protein as a result of an elevation in cell size (23). Studies with ewes indicate a substantial hypertrophy of liver, gut, heart, and mammary tissue (34), which corresponds to the increase in intake (47) and the depression in the weight and protein content of individual muscles during lactation (21). During early lactation, protein content is higher than normal in the liver (33) and lower than usual in skeletal muscle (21), suggesting a restructuring of whole body protein metabolism Indeed, virgin rats have balance of hepatic uptake and output of amino acids, but the onset of lactation induces a high net hepatic uptake (28). Hepatic protein synthesis in mice has been shown to peak just prior to parturition [(104); Table 11 and remains elevated during lactation, although the fractional synthetic rate (FSR) of protein appears to decline with continuing lactation (75, 104). Also, the hepatic extraction of a-amino N in lactating cows is higher than that in steers (71). possibly due to increased hepatic blood flow and substrate availability. During lactation, as during nonlactating periods, diet appears to alter protein synthesis in the liver. Jansen and Hunsaker (75) and Sampson et al. (131) showed a wide range in liver FSR in rats given diets at different levels of intake or diets supplemented with amino acids, and Sampson and Jansen (132) reported lower liver protein absolute synthesis rates in rats fed diets &ficient in protein quality and intake, indicating that actual substrate availability may affect the hepatic protein synthetic rate. Thus, the increased intake during lactation would, in J

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TABLE 1. Effect of pregnancy and Iacmtion on the h- that of the liver. Millican et d. (104) showed tional rate of protein synthesis (FSR) in the liver of mice no significant change in mouse gastrocnemius 8nd rats. FSR

Mouse

Rat

virgin Pregnant 10 d Regnant 18 d Lactating3d Lactating 15 d Casein Lactating 3 d Lactating6d Lactating9d Lactating 15 d wheat gluten Lactating 3 d Ladating6d Ladatbz9d Lactat& 15 d

(%/dl 64.3 65.6 112.0 78.1 73.4 81.8 762 735 58.8 675

(75)

55.6

(75)

58.6 56.9

(75) (75

itself, be expected to Cause enhanced hepatic protein synthesis. Skeletal muscle is the largest protein pool in the body and by its sheer volume contributes substantively to whole body protein turnover and energetics. The role of lactation in skeletal muscle protein metabolism is less clear than

muscle protein synthesis during lactation. They concluded that in the well-fed mouse, in contrast to other species, skeletal muscle was not utilized as a pool or reserve of amino acids during pregnancy or lactation (Table 2). Vincent and Lindsay (157) agreed with the conclusion of Bryant and Smith (21) that skeletal muscles in sheep may undergo a controlled depletion of protein during lactation, but these groups disagreed on the mechanistic basis for this depletion. Using a hind-limb arteriovenous difference technique, Vincent and Lindsay (157) .~ found that lactation induced a 25% increme in protein synthesis, whereas Bryant and Smith (21) reported that skeletal muscle protein synthetic rate was unchanged or depressed during lactation (Table 2). Bryant and Smith (21) were considering different muscle sites, and, as discussed later, individual muscles may have dissimilar metabolism during lactation. Vincent and Lindsay (157) also found a 30% increase in the fractional degradation rate during lactation (Table 2) and indicated that this was the major mechanism for muscle protein depletion during lactation. Individual muscles appear to differ in their FSR and their responses to metabolic alteration.

TABLE 2. Elffects of lactation on the fractional rate of protem synthesis (PSR) and protein degradation (FDR) in skeletal muscle. Species

Physiological state

FSR (Wd)

Mice

Vi@

Regnant 10 d Regnant 18 d

sbaep

shaep

Lactatbg3d Lactating 15 d Lmgissimas dorsi Unmstcd Lactatiqg 16 to 21 d Lactating 56 to 57 d semitendimsis Unmated Lactating 16 to 21 d Lactating 56 to 57 d Nonprtgnant. nonlrtctatios

Rcsnaot

cows

Lactating Lactating controls Lactating + bST

Journal of Dairy Science Vol. 74, No. 2, 1991

FDR

Reference

...

(104) (104) ( 104) (104)

-

4.98 4.96 4.89 455 4 51

... ... ...

3.0 3.2 3.6

... ... ...

3.0 2.1 2.4 2.1 2.8 2.8 3.08 3.39

... ...

...

...

2.1

4.1 3.0

... ...

(104)

(21) (21) (21)

(21) (2 1) (21) (157) (157) (157) McBride (unpublished) McBridc (unpublished)

SYMPOSIUM: NONMAMMARY METABOLISM IN SUPPORT OF LACXATION AND GROWTH

Bryant and Smith (21) indicated that after 16 to 21 d of lactation, the sheep semitendinosus muscle FSR substantially declined, whilst the longissimus value was unafYected. Hunter et al. (70) also repofled different individual muscle changes in growing lambs; the vastus intermedius muscle FSR was higher than either the longissimus dorsi or vastus lateralus in control and estradiol-treate!d lambs. Consistent with this finding is that of Pell and Bates (122), who found that lamb semitendinosus muscles were not affected by Somatotropin treatment, but the bicep femoris FSR was substantially elevated. McBride et al. (97), however, found no change in the in vivo FSR for the sartorius, semitendinosus, or external intemostal muscles of fully grown steers treated with somatotropin (2.39 vs. 2.81; 2.79 vs. 2.85; and 2.56 vs. 3.14, for control and somatotropin-treated animals, Espectively). The individual muscle selected for measurement of protein synthetic rate, as well as its physiological age, appeared to influence the d e t e d a t i o n of protein turnover. Hormonal Influences

Endocrine shifts and alterations during growth and lactation have been shown to have substantial effects on pmtein metabolism (153), and it is reasonable to assume that changes in the rates of protein synthesis and degradation will also alter the amount of energy required to support protein turnover. Maintenance of metabolic homeostasis by endocrine regulation of nutrient partitioning is critical to the lactating animal (14, 129). Early lactation is associated with an energy demand that canuot be satisfied by energy intake, causing altemtions in the concentrations of metabolic regulatory hormones such as somatotropin, glucagon, and insulin (15,121,135) and subsequent mobilization of body reserves (24). Some of the homeonhetic and homeostatic actions of hormones would be expected to modulate the metabolism of proteins during the lactational state (4) and would therefore influence energy expenditure on protein turnover. Circulating plasma somatotropin, which is higher in lactating as opposed to nonlactatjng cows (133). increases milk production and irrs versible losses of glucose in lactating dairy cows (9). even when these animals are in negative energy and nitrogen balance (149). After

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treatment with exogenous somatotropin, cows tend to be in even more negative nitrogen balance (149). Milk production is modulated by homeorrhetic shifts sparked by the metabolic state of the animal in order to maintain glucose homeostasis (95). Somatotropin may reduce tissue responsiveness to insulin, a primary anabolic hormone (8), and redirect glucose elsewhere, possibly to the mammary gland In vitro, increased rates of amino acid uptake and protein synthesis appear to be due to the insulin-like action of somatotropin (25). Somatotropin treatment enhances foregut tissue metabolism and growth via increased cellular hypemphy (20). facilitating enhanced nutrient absorption. Furthermore, Somatotropin has been shown to increase skeletal muscle protein synthesis in lambs with no change in degradation (122). Indeed, in growing animals, elevated nitrogen retention in steers (44) and sheep (41,122) has been attributed to increased protein synthesis rather than decreased protein degradation. Thyroxine ("4) and triiodothyronine ("3) levels in plasma both decline throughout the course of lactation (148). Milk production and plasma T3 concentrations are inversely correlated (151), whereas other hormones such as glucagon and somatotropin are elevated (148). During a thyroid deficiency, muscle proteolysis is depressed (57). 'Ihyroidectomized rats show depressions in muscle protein synthesis and degradation (108). This may be a generalized response during a muscle-wasting state. This is also the case in the diabetic condition, in which there is depressed plasma T3 concentration and muscle proteolysis. A similar response is also seen in cold-stressed animals in which plasma T3,feed intake, and muscle proteolysis are all increased (86, 147). In sheep, McBride and Early (96) found an increase in the fractional rate of skeletal muscle protein synthesis when treated with T3. Indeed, T3 may help mediate the changes in the rates of protein synthesis and degradation and thereby support metabolic regulation of protein turnover. The specific role of insulin in maintenance of lactation is not clear (136), although circulating plasma levels are negatively correlated with milk fat percentage and milk yield (15, 67). Postpartum,metabolism in the lactating rat appears to be highly regulated by both plasma J

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insulin and glucagon concentrations (162). During the early lactation of d a i q cows, the insulin:glucagon ratio is depressed (133), and even though insulin levels increase after 4 wk of lactation (85), they may still be lower than for nonlactating animals (133). Serum insulin rises steadily fmm 50 to 300 d of lactation (67) and appears to be inversely related to milk production. Insulin levels are highly responsive to plane of nutrition. Feed restriction is associated with depressed serum insulin levels in lactating sheep (58). Serum insulin concentrations can be made lower in high yielding cows through underfeeding. This is not the case if nutrient

intake relative to their requirement is equal in both the high and low yielding cow (64). Increased hepatic glucose output is promoted by decreased circulating insulin in ruminants (67). even though the number of insulin receptors in hepatocytes does not change during lactation. The plasma insulin:glucagon ratio is depressed during lactation, enhancing circulating plasma glucose concentration and availability to the high demands of the mammary gland. TRANSPORT OF SODIUM AND POTASSIUM ION

Water and salt intakes by animals are periodic, but the transmembrane Na+ electrochemical gradient and intracellular and extracellular osmolarity must be maintained within narrow limits at all times in order to achieve nutrient TABLE 3. Ouabain-sensitive respiration in various ovine transport, nerve system function, enzyme activtissues. ity and cell volume in mammals (106, 144). Milligan (105) hypothesized that Na+,K+transouabainport across the plasma membrane, an essential physiological sensitive Time state remiration Reference process maintaining the Na+ gradient, may account for a major part of the metabolic component of maintenance energy expenditure in the Duodenal animal. Recently, Gill et al. (54)concluded that Starvation mumsa 28.6 LOW DE intake' 48.1 Na+, K+ transport accounted for 16 to 20% of High DE intake 61.3 whole body aerobic energy utilization in young 20.5 Liver Fetal, 75 d old sheep. Swaminathan et al. (143) also supported 11.5 Fetal, 97 d old the previous hypothesis showing Na+, K+ trans12.4 Petal, 136 d old Starvation 17.8 port contributed about 40% of the resting 0 2 45.8 Peak lactation consumption in vivo in the guinea pig. 36.5 Nonlactating During the last 10 yr, however, the results in Thyroidectomy 18 our laboratory have indicated that the amounts T h p i k t o m y + T3' 24.2 of Na+, K+ transport varied with different tisHepatocytes 1 wk old 52.5 3 wk old 54.3 sues, physiological state, and nutritional condiLamb 47.8 (106, 107, 142). Tables 3 and 4 illustrate tion Saline 28.6 wide variance in levels of inhibition of 02 the + T41 35.7 consumption by ouabain [a specific inhibitor of Muscle Thennoneutral 28.5 Cold 45.0 Na+, K+-ATPase; (56)] in sheep and cattle. As 18.1 saline noted earlier, lactation triggers broad metabolic 26.5 + T4 changes, especially nutrient utilization in variPlacenta Maternal ous tissues. Moe (110) indicated that lactating Tbtrmoncutral 20.7 Heat 18.5 cows have a 20% higher maintenance energy Fetal requirement than nonlactating cows. Gregg and ThermoneuIral 29 Milligan (59) illustrated that energy expended Heat 27.3 on Na+, K+-ATF'ase by the sternomandibularis Parotid gland In vivo 66.5 muscle from nonlactating ewes required 42% of In vim 41.0 total 02 uptake, but this proportion was 46%, Whole bodv Modeled 16 to 20 and total aerobic energy expenditure on 'DE = Digestible energy, T3 = biiodothyronint, T4 = Na+,K+- ATPase was highly increased in lactating ewes. Furthermore, McBride and Milligan thpxhe. J o d of Dairy Science Vol. 74, No. 2, 1991

SYMPOSIUM: NONMAMMARY METABOLISM IN SUPPORT OF LACTATION AND GROWTH

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TABLE 4.Ouabain-sensitive respiration in various bovine tissues. ouabainPhysiological TiSSUe

sensitive remiration

State

Reference

(%)

Rumen epithelium Grass hay Duodenal mucosa Nonlactaling Peak lactation Late lactation Late lactation + chronic Liver Control + bST Muscle 10 to 21 d old 7 mo old

ST'

16 to 19 34 to 35 55 31.1 21.6 16.1 21.1 41.3 39.4

'somatotropin.

(98) demonstrated that 0, consumption on Na+,K+-ATF'ase activity accounted for 55% of total mucosal respiration of peak lactation cows, but after lactation the proportion of ouabain-sensitive 0, uptake declined to 35%. The high proportion of Na+,K+-ATPasedependent respiration in intestinal mucosa of lactating cows may be related to hyperplasia or hypertrophy due to higher feed intake (47,48). Similarly, h c h k e et al. (91) reported that Na+,K+ATPase activity per unit area, weight, or p r e tein content was increased by up to 70% over that of control animals in hypertrophying rat cecal mucosa induced by polyethylene glycol (PEG) feeding, whereas other enzymes (glutamate dehydrogenase, alkaline phosphatase, MPase, and lactate dehydrogenase) remained unaltered. The amount of energy spent on Na+,K+ATPase may be influenced by the level of feed intake in animals. In mice overfed sucrose, Na+,K+-ATPase activity in liver and skeletal muscle increased by 88 and 26%, respectively (50). The results from our laboratory have shown that energy expenditure for Na+,K+ATPase activity of duodenal mucosa increased with the level of feed intake of sheep (99).That study indicated that ouabain-sensitive respiration of duodenal mucosa in the higher fed group accounted for 61% of the total 0 2 uptakes, but the proportions in the lower or 48-h fasted groups showed 48 or 29%, respectively. Changes in Na+,K+-ATPase-relatedenergy costs resulting from an alteration in plane of nutrition, diet volume, or the lactational state may result from a common mechanism of mod-

ified nutrient supply and uptake from the GIT. Whether increased nutrient intake (and perhaps increased dietary volume) produces parallel effects on pump activity in lactating and growing animals is unclear, but it is apparent that the lactational state and the level of intake in growing animals can both produce very major shifts in ouabain-sensitive respiration. McBride and Milligan (100) measured elevated Na+,K+-AWasedepndent respiration in liver biopsies excised from lactating ewes. Ouabain-sensitive 0 2 consumption in peak lactation accounted for 45% of the total 0 2 consumption of the liver biopsy, but this value was 37% for the dry period. Milligan and McBride (106) assumed that the increased activity of Na+,K+-AWase in skeletal muscle, liver, and gut mucosa of lactating animals is related to the endocrine changes occurring in support of lactation. More specifically, the increased amount of Na+,K+-AWasedependentrespiration in the gut mucosa of lactating animals is probably related to elevated feed intake and subsequent increased cellular proliferation and differentiation during lactation. Recently, Bergstrom and Norrby (11) suggested that epidermal growth factor (EGF)might be involved in intestinal cell production during lactation, based on an observation of increased plasma EGF concentration during lactation (158). Consequently, intestinal tissue during lactation might be controlled by the successive metabolic events triggered by EGF that were described by Rozengurt (130): 1) mitogenic stimulation, 2) enhanced amilonde-sensitiveNa+,H+ exchange, 3) secondary stimulation of Na+,K+-ATPasebeJournal of Dahy Science Vol. 74, No. 2, 1991

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TABLE 5. Effects of physiologhl state on triacylglycerokfrcc fatty acid cycling. SQeCicS

Physiological state

Tissue

cvclinn rate

Reference

control)

(%I

Mouse Mouse Mouse Mouse Mouse Mouse Rat Rat HllLllEM

Goat

Goat

White adipose White adipose White adipose White adipose Brown adipose Brown adipose Fpididymd fat Epididymd fat Whole body Whole body Whole body

Starved, 24 h Wad, 50 m@g Ttiiodothyronioe, 500 ClglLg Cold exposure

53 509 176 116

4 -

284

50 m@g

208

Cold exposure Glacagon. 5

289

A5 P@ Starved, 4 d

669

Ladatin& 38

w

d hctathg. 76 d

228 2331 3951

lControl rate. tslren as cycling at 10 d of lactation.

cause of intracellular Na+ accumulation, 4) induction of cytoplasmic alkahzation by Na+,H+ exchange, and 5) initiation of DNA synthesis and cell division. "

SUBSTRATE CYCLES

Further control of cellular me€abolism during growth and lactation is achieved by the regulation of substrate fluxes through specific biochemical pathways. When a nonequjlibrium reaction sequence in the forward direction of a pathway is opposed by another nonequilibrium reaction sequence in the reverse and one of the reactions requires an energy input, a substrate cycle may exist. Such cycles allow for very sensitive and flexible direction control, but in the earlier literatme great attention was focused on the hydrolysis of ATP to ADP or AMP and inorganic phosphate in substrate cycles, and they were described as futile cycles. The ATP hydrolysis aspect of substrate cycles could fit them for a role in the regulation of thermogene sis and in control of BW (83, 113, 115). For a thorough review of the mechanism of metabolic control by substrate cycles, the reader should refer to Crabtree and Newsholme (37). Some examples of substrate cycles operating in aaimal tissues are those involving fructose 6-phosphate (F6P) and fructose l,&biSphosphate (FBP) in glycolysis, triacylglycerol (TG) and FFA in lipid tumover, glutamine and glutamate in amamnia detoxification. and the Cori cycle in muscle lactosis. JOprnal

of Dairy Scicncc VoL 74, No. 2, 1991

The quantitative role and associated energy cost of each substrate cycle appears to depend on the metabolic functions of the tissue or organ considered. Thus, in liver, which must respand to short-term changes in nutrient supply and sups high capacities for both glycolysis and gluconeogenesis, 21 to 26% of total ATP expenditure is used by substrate cycles involving glycolysis and the acetate-acetyl coenzyme A (AcCoA) pair (125). However, in skeletal muscle, a nongluconeogenic tissue, F6P-FBP cycling accounts for only .5% of in vitro energy expenditure (30), whereas the comparable cost for hepatocytes in vitro is 9 to 14%. Triacylglycerol-Free Fatty Acids

This cycle has been studied in white and brown adipose tissue (7, 18, 19) and in the whole body (45). The operation of the TG-FFA cycle brings about hydrolysis of 8 ATP to 8 ADP per turn (114). In rodents, researchers at Oxford (18, 19) demonstrated very substantial hormonal effects on the rate of this cycle in white and brown adipose tissue. Catecholamines, T3, adrenocorticotropin, and glucagon, but not insulin, all altered the rate of cycling (Table 5). Addition of insulin together with adrenaline to the rat epididymal fat pad potentiated the effect of the catecholamine on FFA reesterification such that cycling was eightfold greater than in control (no hormone) samples (7).In vivo increases of fivefold in the TG-FFA cycle have been produced by treatment of the

S y M p o s N M : NONMAMMARY METABOLISM

animal with p-agonists such as fenterol (19).

The influence of pharmacological doses of hormones on a substrate cycle may not truly reflect the physiologic adaptability of that cycle. However, starvation for 24 h was shown to halve the cycling rate of TG-FFA in white adipose tissue from mice, whereas acute cold exposure (4 h) doubled the rate of FFA resynthesis in brown adipose tissue (19). Very recently, Dunshea and Bell (43) showed that FFA recycling increased from early to later lactation in Saanen dairy goats such that the TG-FFA Cycling rate was four times greater at 76 d of lactation than it was at 10 d postpartum. Functionally, the low rate of FFA rwsterification in early lactation likely related to the depressed voluntary intake and high metabolic demand for milk (and milk fat) production. Simultaneous measurement of the rates of 02 consumption and FFA reesterifkation in adipose tissue suggests that a close relationship exists between energy usage by the TGFFA cycle and total heat production of this tissue (7). In the well-fed rat, about 2% of resting metabolic rate (RMR) may be attributed to the TG-FFA cycle, rising to as much as 15% of RMR during hormonal challenge (7). In humans, after an overnight fast .4% of RMR is associated with the TG-FFA cycle, increasing to 2.5% after 4 d of starvation (45). These experimental values are in agreement with Baldwin and Smith (5), who calculated a mean energy cost for FFA reesterification of 1.2% RMR for humans. Estimates for young (30 kg) pigs suggested that 3.9% of RMR was due to the cycling of TG-FFA (128), which was within the range of 2 to 4% RMR given by Baldwin et al. (6) as the cost of lipid resynthe sis in growing or lactating animals. consistently then, the "G-FFA cycle constitutes only a small portion of RMR (.4 to 4%), even in the midlactation animal (A. W. Bell, personal communication). Chadzidukis et al. (29) proposed that a depression in the rate of the TG-FFA cycle may account for the excessive fat deposition in Zucker obese rats contrasted with lean rats. In our laboratory, we are presently searching for a metabolic explanation for the variation in growth rate within a population of broiler chicks. The abdominal fat pad in fast growing chicks of 7 d of age is 44% larger per unit of BW than in slow growing chicks. This may be

687

IN SUPPORT OF LACTATION AND GROWTH

attributable to a significant reduction in the rate of FFA recycling in fast growing birds Uable 6). Also, the TG-FFA cycle in avian adipose tissue apparently is not sensitive to the effects of T3, A m and noradrenaline, which are known effectors of FFA reesterification in mammals (18, 19). However, glucagon does modulate the activity of the cycle, which is not surprising because this hormone has been suggested as the major regulator of lipolysis in birds (87). Glycolysls and Gluconeogenesls

The four identified substrate cycles in the glycolytic pathway are glucose-glucose 6-phosphate (GL-G6P), glycogen-G6P, F%P-FBP,and phosphoenolpyruvate - pyruvate - oxaloacetate (PJ3-Pyr-OAA). In addition, there is an acetateAcCoA pair linking to the tricarboxylic

TABLE 6. Release QmoVg DM/h) of glycerol and FFA and t h ~ ~ l g - 1 cycling (clmoug DWU in thc abdominal fat pad of fast (F) and slow (S) growing week-old bila chicks as inflacnctd by somt hormoms. HormOne

Glycerol FPA release release

CLCk

Control

P

198 157

-72 173

153 167

15

94 108

188 148

94 176

249b 310

415b 571'

3ma 390

a5 166

225 254

30 243

123 179 124

241 257 136

371

chick Growth rate Bird x grrnvth rate

NS

.01 NS NS

Hormoot

.MI1

78 191

S

Insulin

P S Triiodothyronine

F S

56 5gp

a

Glucap

P S

Adrenocorticotmpin P S

Noradrenalim

P S

SD Main effects

.05

NS

140

aa NS .05

NS

.MI1

.05

NS

NS

Joranal of Dairy Scieace Vol. 74, No. 2, 1991

688

KELLY ET AL

TABLE 7. Effects of glucagon on substrate cycling. Cycling rate Species

Tissue

Cycle’

with glucagon

Reference

(96 control) Rat Rat Rat Rat Rat Rat chick

Hepatocytes Hepatocytes Hepatocytes Hepatocytes Hepatocytes Epididymal fat pad Abdominal fat pad

GGG6P GlycogenG6P F6P-FBP PEP-Pyr-oAA Acetate-AcCOA TG-FFA TG-FFA

23 33 75 58 104 289 69 1

(125) (12.3

(125) (125) (125)

(18) summers and Milligan

(UnPaMished)

‘GL = Glucose, G6P = glucose 6-phosphate, F6P = fructose 6-phosphate. FBP = f ~ ~ c t o s1,6bisphosphate, e PEP = phosphoenolpyruvate, qrr=pyrUvate,OAA = oxaloacetate, AcCoA = acetyl coenzyme A, TG = triacylglycml, FPA = free fatty acid.

acid cycle. Recently, in a very carefully controlled study, 21 to 26% of ATP expenditure by isolated rat hepatocytes were used by the substrate cycles involving glycolysis and the a c e tate-AcCoA pair and that glucagon treatment differentially altered the rate of the cycles [(125); Table 71. Quantitatively, the F6P-FBP cycle was the most energetically costly cycle accounting for 8.8 to 14.2% of ATP consumption in hepatocytes (142). In bumblebee species, which feed by foraging on “masses” of flowers, nonshivering thermogenesis resulting from the F6P-FBP cycle is thought to control endothermy during periods between flight (116, 124). Clearly, the F6P-FBP cycle is a major component of energy costs in bees. In rat skeletal muscle, up to 13-fold increases were found in the F6P-FBP cycling rate in response to adrenaline and various j3agonists (30, 31, 32). Such marked changes in one substrate cycle may produce only a small rise in total heat production by a tissue, but if several cycles respond in parallel to a stimulus (e.g., catecholamines), these changes may account for a considerable portion of the total thermogenic response to that stimulus (32). Treatment of hepatocytes with glucagon, for example, reduces cycling at the four substrate cycle sites in glycolysis [(125); Table 71. The metabolic role of the acetateAcCoA cycle is at present unclear, although in rat hepatocytes at least it would appear not be increase the sensitivity of mgdation of acetate utilization to blood acetate concentration and has only a minor thermogenic role (77, 125). In Journal of Dairy Science Vol. 74, No. 2, 1991

ruminants, however, because of the emphasized acetate metabolism and the high endogenous entry rate of acetate (lo), the possibility of a substantial energy cost for the acetate-AcCoA cycle is particularly pertinent. Other Substrate Cycles

Newsholme (114) provided a comprehensive list of substrate cycles that might be important in thermogenesis and protein, carbohydrate, and lipid turnover, plus ion translocation. Several unique substrate cycles are also being discovered, such as deoxycytidinedeoxycytidine 5’-phosphate in B lymphoblasts (but not T lymphoblasts) and in fibroblasts (13, 73) and an AMP-adenosine cycle in hepatocytes (17). Within the whole animal exist numerous organ pools; several metabolite cycles have been identified whereby substrates are shifted from one organ to another and back again (often in a different biochemical fom) with a resultant interorgan metabolite transfer. For example, large quantities of alanine and glutamine are released from skeletal muscle, especially during fasting, and are subsequently taken up by the splanchnic tissues or kidney. Much of the glutamine metabolized in the gut and kidneys forms alanine, which passes to the liver where it is a substrate for gluconeogenesis (117). The GL thus fomed enters the peripheral circulation from where it is taken up and used by skeletal muscle. An alanine-GL cycle results. Similar cycling occurs between lactate and GL (Con cycle) and accounts for 2% of energy expenditure in growing pigs (128). En-

SYMPOSlUht NONMAMMARY METABOLISM IN SUPPORT OP LACTATION AND GROWTH

ergy use in both of these cycles is linked to the oxidation of fat in liver and to the secondary active transport of the substrate or product across the cell membrane. Stangassinger and Giesecke (140) reviewed data on the combined Cori and alanine cycle activities in ruminants under various physiological circumstances. In fed or GL-loaded animals such cycling is minimal, however, it increases markedly after prolonged (72 h) fasting and is further intensified when the fasted animal receives phloridzin, a Na+-GL transport inhibitor. During pregnancy and lactation, recycling appears to become enh a n d such that the combined con plus alanine cycles account for 10 to 20% of the total GL production rate.

689

events is widely different. CONCLUSION

Some direct data and a good deal of inferential material have been assembled fkom which lactation and growth appear to entail substantial changes in principal metabolic processes involving maintenance. These processes include active ion transport across cell membranes (particularly the transport of Na+ and K+),protein turnover, and substrak cycles. It is heartening that several studies are now beiig directed at achieving quantitative in vivo measurements of metabolic conversions of specific tissues and changes therein with physiological state. If for no better reason than the need to cope with processing and absorption of the increased METABOLIC REGULATION nutrients, researchers are focusing on the abdominal organs and particularly the GIT as a Metabolic control is achieved at the expense site of considerable adaptation in the productive of energy. This is most evident at the cell state. Widespread metabolic change is likely membrane, where transport processes (Na+,K+- necessary to achieve and support productive ATPase, Ca2+-ATPase,and Na+, Mg+-ATPase) functions. To understand such metabolic interall function at the cost of high energy phos- action fully mechanistic metabolic models phate bonds. Further regulation is also shown would be beneficial, if not necessary. These within the cell membrane through intramem- models would also lead to more precise nutribrane transduction. An example of this was tional management. provided by Bemdge (12) where the G-protein cycle was described. After stimulation by an REFERENCES external signal (for example, epinephrine on plAmenomori, Y., C. L. Chen, and J. Meites. 1970. adrenergic receptors on skeletal muscle cells), Serum prolactin levels in rats during reproductive the G-protein is transmitted through the cell states. E!ndocrinology 86:506. membrane, where it becomes available for 2 A O YY.. ~ I. T d , J. I. Okumura, and T. M-tbinding by guanosine triphosphate (GTP) (the su. 1988. Energy cost of whole-body protein synthesis measured in vivo in chick. Comp. Biochem. Physiol. “on” reaction). This process facilitates the G91:765. protein-GTP complex stimulation of adenylate 3 Ardawi, MS.M 1987. The ‘ 1activity of phoscyclase and the subsequent formation of cyclic phate-dependent glutamhue and glutamine metabothus, the message from the initial signal lism in late-pregnant and peak-lactating rats. Biockm. J. 24275. is passed on. Once this has occurred, the G4Baird, G. D. 1981. Lactation, pregnancy and metaprotein-GTP complex is terminated via the hybolic disordex in the ruminant. Proc. Nutr. SOC. 40: drolysis of GTP to guanosine diphosphate 115. SBaIdwin, R L., and N. E. Smith. 1974. Molecular (GDP) [the “off” reaction; (12)]. This concept control of enmgy metabolism. Page 17 in The control may be extended to even lower levels of orof metabolism. J. D. Sink, ed. Pennsylvania State ganization of intracellular metabolic control in Univ. Press, Univa-sity Park that events such as protein turnover (127) or 6 Baldwin, R L., N. E. Smith, J. Taylor, and M.Sharp. 1980. Manipulating metabolic parameters to improve substrate cycling (18, 19, 125) are subject to growthrateand milk secretion I. Anim. Sci. 513416. endocrine manipulation. The whole process of 7Ball, E. G. 1965. Some amgy relationships in adimetabolic control and energy transduction is, pose tissue. AWL NY Acad. Sci. 131:225. therefore, consistent through the different levels 8Bauam. D. E., and S. N. McCutcheon. 1986. The effects of growth hormone and prolacbh on m e t a b of cellular organization (e.g., at the membrane; lism. F%ge436 in Control of digestion and m e t a b within the cell membrane; and finally within lisminruminants. L. P. Milligan, W. L. Grovum, and the cell and cellular organelles). However, the A. Dobson, ed. Prentice-Hall, E~~glewood Cliffs. NJ. magnitude of energy directed to these control 9Bamnan, D. E., C. J. Peel, W. D. Steinbur. P. J. J o d of Dairy Science Vol. 74, No. 2, 1991

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