Regulation of Adipose Tissue Metabolism in Support of Lactation' J. P. McNAMARA Washington State University Department of Animal Science pullman 991M-6320 ABSTRACT

In the dairy cow, adipose tissue lipid accumulates during pregnancy, and catabolism begins prior to parturition and increases dramatically afterward. After peak lactation, body lipid is replenished. The duration and magnitudes of these adaptations depend on milk energy secretion, net energy intake, genotype, and endocrine environment. Recent research efforts have focused on endocrine, genetic, and biochemical mechanisms underlying metabolic adaptations in cows of high production potential. Adipose tissue lipid synthesis is decreased and lipolysis is increased in early lactation. The magnitude and duration of these adaptations are increased in animals either consuming relatively less energy or producing more milk. Adipose tissue is more responsive to catecholamines in early and midlactation and in animals with higher production. This is more of an increase in maximal response than in sensitivity. In vivo and in vitro rates of adipose tissue lipolysis correlate positively with milk energy secretion, whereas lipid synthesis rates correlate with energy intake. Thus, mammary metabolic activity, within and among lactations, correlates with that in adipose tissue. Likely mechanisms include adaptations in receptors for homeostatic signals and modulation of postreceptor responses. Research is needed into neural, genetic, and hormone regulation of nutrient utilization and body

fat use and recovery during lactation. Research should describe mechanistic relationships among nutrients in animals of high production as well as investigate cellular and molecular mechanisms suitable to genetic manipulation. (Key words: adipose, lactation, metabolism)

Abbreviation key: CoA = coenzyme A, E = epinephrine, HSL = hormone-sensitive lipase activity, Km = Michaelis constant, LPL = lipoprotein lipase, NE = norepinephrine, SNS = sympathetic nervous system, ,,V = maximum velocity. INTRODUCTION

Adipose tissue functions to provide energy in the form of fatty acids and glycerol to other organs, especially when the dietary intake does not meet requirements. During lactation, the major portion of this energy is used by the mammary gland. Thus, adipose tissue helps provide the proper metabolite mixture to the mammary gland A subfunction is to synthesize fatty acids for release to the mammary gland as needed to maintain milk fat secretion. The contribution of these functions to the energy economy of the animal is highly variable and is affected by many factors: dietary energy and fat intake, rate of milk synthesis and milk fat secretion, maintenance energy requirements, and energy balance. Adipose tissue in mammals is highly regulated. Adipose tissue may have been developed to support the physiologic states of pregnancy and lactation in order to overcome periods of energy and fat deficit so that nourishment of the young could be ensured (51). A primary Received August 21, 1989. Accepted January 29, 1990. purpose of study in this afea is to quantify the 'Papa N m k 8003. College of Agriculture and Home metabolic pathways and regulatory mechanisms Economiar Research Center, Washington State University, that are essential in providing the proper pullmaa Project Numbex 0663. Supporwd in part by MH amount and mixture of metabolites to the mamGrant HD-2452941. 1991 J Dairy Sci 747W719

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mary gland during the various stages of lactation. This is especially important to the nutritional management of high producing dairy animals. This review is not comprehensive. The reader is directed to the work of other researchers for more detailed background information (1, 2,3,4,5, 6,7, 8, 18, 19,21,22,27, 35,50, 70, 73, 77). This review describes basic facts and concepts and provides further detail on con~butionswithin the last several years: advances made in defining the relationships of adipose metabolism to whole animal energy inputs and outputs, new definitions of mechanistic regulatory adaptations, and refinements of mechanistic deterministic models of dairy cow metabolism. A primary purpose of the review will be to point out (when data are available) and to speculate on (when data are not available) the relationship of adipose tissue metabolism to production and efficiency of the lactating animal and to present hypotheses for fruitful new lines of research. DISCUSSION Adipose Tissue Metabolism During Lactation

In 1980, Vernon (69) reviewed the literature on lipid metabolism in ruminant animals and stated 'Ihe above summary, however, is based on a very limikdnumber of observations; there is clearly a need for more systematic studies of adipose tissue m e t a b o m during pregnancy and lactatioa with attention to such variables as number of foetuses, milk production and energy balance. It is perhaps surprising that no real attempt has been made to collate adipose tissue metabolism with capacity for milk production.

This lack of attention was not due to low scientific interest or importance, but rather to the need for available suitable animals, techniques, and research funding. Adipose tissue of the pregnant female was already known to go through a period of lipid accumulation through part of gestation. Toward the end of gestation, at a time and of a magnitude dependent on the species, the dominant state becomes catabolism. This is accompanied by rapid loss of stored lipid in early lactation followed by (again species dependent) a sustained

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catabolism during lactation. Either at weaning or after peak lactation, anabolism again becomes the dominant state for maternal recovery. In the cow, the initial metabolic adaptations OCCUT betwm about 30 d prepartum and parturition. After parturition, catabolism increases and anabolism decreases for a period dependent on genotype and environment. These adaptations are not isolated biochemical events but rather a well-orchestrated resetting of cellular structure and function. In cows, this is manifested as a decrease of lipogenesis and esterification accompanied by an increase of lipolysis. The initial large flux of lipid from the adipose tissue in the first 14 to 21 d of lactation appears to be the summary result of all three processes (1,2, 5,7, 8,29,43,44,45,46, 47,48, 57, 58, 59, 61, 71, 72, 73, 78, 83, 84). The uptake of preformed lipid via lipoprotein lipase &PL) may also be decreased during the peripartum period (57). However, the contribution of this pathway to adipose lipid flux is s t i l l not defined in the bovine. The best estimates are that this pathway accounts for approximately 18% of the total fatty acid uptake (27). During the first several weeks of lactation, adipose tissue is adapting its biochemical structure and function to the lactational state. During this time, the lipolytic pathways increase in activity. This is manifested as an increase in basal lipolysis, measured by fatty acid and glycerol release in vitro, hormone-sensitive lipase activity (HSL) in vitro (43,44,45,46,47, 48, 83, 84), and by increased fatty acid and glycerol concentrations in blood (58, 59). The lipolytic responsiveness to hormones and neurotransmitters is altered as well. The tissue (27, 45, 47, 48, 83, 84) and the animal as a whole (58,59) are more responsive to catecholamines, which bind to fbadrenergic receptors to elicit an increase in cyclic (c) AMP. This causes an increase in cAMP-dependent protein kinase, which phosphorylates HSL, thus increasing the rate of lipolysis. Key questions are how the in vitro measurements, especially of lipolysis, relate to the actual in vivo lipid fluxes at each stage of lactation and at different milk yields and how such information might be used to predict performance. Although these cell adaptations have been identified, their relationship to the whole body lipid utilization is only beginning to be quantified. However, a useful Journal of Dairy Science Vol. 74, No. 2, 1991

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TABLE 1. Summary of genetic and dietary effects on adipose metabolism in bovine lactation' Physiological state Early lactation

Late pregnancy

Adipose function Geneticmerit Energy

Adaptation Higher

Higher

Increases

Basal lipolysis

Basal lipolysis

Stimulated

Stimulated

lipolysis HSL~ Lipogenesis Esterification

lipolysis HSL Lipogenesis EsteScation

Decreases

Support of lactation

Fat cell size

Higher Lipgenesis estesification Fat cell Size

Late lactation

Support and recovery Higher

Basal lipolysis Stimulated lipolysis

Higher Lipogenesis esterification Fat cell size

LPL3

Basal lipolysis

Fat cell size

Stimulated lipolysis HSL

'All data derived from published in vitro adipose tissue incubations and enzyme assays (41, 42, 43. 44, 45, 46). 'HSL = Homnssensitive lipase. 3Lm, = Lipoprotein lipase.

mechanistic model of dairy cow metabolism, including lipid metabolism, had been constructed 10 yr ago using in vitro kinetic data [maximum velocity (V& and the Michaelis constant (Kd]of the key pathways, available in vivo data, and sound biological assumptions. These research fidings led to specific attempts to define the timing, magnitude, rate, and regulation of the myriad lipid metabolism adaptations. In addition, integrating data into a mechanistic model system required more specific kinetic data than were available. With this in mind, researchers at Washington State University began a study into the lipid metabolism of dairy animals. The model included genetic selection and dietary energy as treatments expected to alter lipid metabolism. Two lines of cows, daughters of high PD milk or average PD milk bulls, were challenged with either a high or low energy diet for the first 140 d of first lactation (44). To date, six sets of first generation heifers (each set comprised daughters of different bulls) have averaged 8863 kg and 7500 kg of milk (305 d) for the high genetic (HG) and low genetic (LG) line over both rations. The HG animals fed high (adequate) energy 0averaged over 10,909 kg of mature equivalent milk per 305 d. Between the two rations, there was approximately a 4 kg/d difference in milk yield, no difference in milk energy secretion, and a 10 McaVd difference in calculated net energy balance (measured from Journal of Dairy Science Vol. 74, No. 2, 1991

28 to 140 d). Thus, a set of animals ranging from 25 to 103 kg/d milk production and -16 to 10 McaVd net energy balance were available for a study of the relationships of adipose tissue metabolism to whole body energy uses. Subcutaneous adipose tissue was biopsied twice prepartum and 6 to 8 times postpartum, covering most of lactation (43,44). In vitro rates of lipogenesis from acetate, esterification from palmitate, FFA and glycerol release, epinephrine (E) and norepinephrine (NE) challenge, LPL and HSL activity, and adipose tissue cellularity were determined. A brief summary of qualitative iindings are given in Table 1. In general, lipogenesis decreased greatly by 15 d postpartum with a smaller drop in esterification (42,43,44,46),which confirmed previous work done with older cows (7, 27, 48). This decrease was detectable between 30 and 15 d prepartum (43). Lipolysis rates, both basal-stimulated and catecholamine-stimulated, increased at the same time (44). A rise in lipolytic activity could be detected prior to parturition and prior to changes in fat cell size (44,62).However, in contrast to previous work (57), activity of LPL was not changed between 30 d prepartum and 30 d postpartum (46). Previous work in primiparous bovine animals detected a decrease in LPL of approximately 30 to 40% (57), but this was determined over a longer period: from 90 d prepartum to 14 d

SYMWSIUM: NONMAMMARY METABOLISM IN SUPPORT OP LACTATION AND GROWTH

postpartum. The accumulated data do not s u p port the hypothesis that LPL is the primary contributor to the decrease in adipose lipid stores in primiparous bovine animals, but it does not rule out a larger role in higher producing, higher parity animals. Similar adaptations were demonstrated in omental and subcutaneous adipose tissue of multiparous dairy cattle (61), including a decrease in lipogenesis, fatty acid synthetase and supporting dehydrogenases, and in LPL. These results confirm those found in our laboratory. However, the large decrease (greater than 80%) in LPL at 5 to 6 wk does not agree with previous work (45,57). Such a decrease in LPL can be explained by the negative energy balance (-6 McaVd) calculated from results in those animals (61). The heifers used in our study ate more and were in greater energy balance (46). Again, there are adaptations due to lactation and further adaptations due to individual animal attributes and environments. Lipolysis was found to have a more complex nature during midlactation. Both basal and stimulated rates of in vitro lipolysis continued to increase during this period, reaching a peak at 120 or even 180 d in some animals (42, 43, 46). This was also true for the maximal response of glycerol release to graded doses of E, such that the V, for glycerol release in response to E was highest at 120 d (41). The V, of FFA release to E peaked at approximately 30 d, 90 d prior to that for glycerol. This confirms the early work of Metz and Van den Bergh (48). who determined that reesterification is lowest at 30 d, even though lipolysis remained elevated past that time. Thus, the net flux of FFA from the tissue was greatest at approximately 30 d, but the actual triglyceride hydrolysis reaction, as measured by glycerol release, remained at fast rates. These findings are consistent with the hypothesis that adipose tissue lipolysis is under continual hormonal or neural stimulus for increased CAMP from parturition until at least 180 d (46). These signals may include release of NE from the sympathetic nervous system (SNS), diminished insulin or elevated somatotropin, E from the adrenals, and possibly thyroid hormones. After peak lactation, cows meet most of their energy needs with feed intake; thus, adipose tissue no longer needs to supply net amounts of energy to the mammary gland. 'Ihe

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actual rates of loss and renewal of body fat are still unknown for high producing dairy cattle (31). Not surprisingly, recent research has determined that adipose lipid metabolism is most variable, and highly related to energy inputs and outputs, in the period of approximately 30 to 180 d of lactation (43, 44, 45, 46). In sheep and goats, the same adaptations take place, with a decrease in activity of enzymes catalyzing anabolic pathways from pregnancy to lactation (69, 71, 73, 78, 79). Similar results were found in studies of whole animal metabolite flux (23, 24, 25, 26). Fatty acid entry and irreversible loss rates were higher in lactating goats, and glycerol entry rate was also higher (26). These in vivo studies are consistent with the in vitro ones and can be more useful in relating to animal energy and carbon inputs and outputs. The activity of adipose tissue HSL (measured at optimal conditions in tissue cytoplasm) peaks at 30 to 60 d and remains elevated until 240 d (46, 62). This pattern is parallel, but only qualitatively, to that of glycerol release from tissue slices, because HSL activity did not continue to increase during midlactation. In a later study with similar animals, HSL activity did decrease after peak lactation, but it remained higher than prepartum (62). This raises a number of interesting possibilities. One is that HSL measured in in vitro homogenates at saturating substrate concentration is only a crude indicator of the rate of the in vivo reaction. This could be because of destruction of the normal intracellular structure or because of removal of the intact phosphorylation system. Another possibility is that the amount of HSL expressed at 30 d and beyond is sufficient to account for the continued increase in tissue glycerol release. This is possible because 1) HSL has a very fast catalytic rate (11, 12). such that the rate in an in vitro optimized system may not truly estimate the maximum rate in a structurally intact cell [this is consistent with the fact that HSL per gram of tissue measured in homogenates routinely gives rates of activity approximately onehalf of the corresponding basal rate in tissue slices (43,44, 45.46, 62)]; and 2) tissue pieces may still be under endogenous regulatory influences when incubated immediately after removal from the animals, and this influence is lost in freezing and homogenizing or even in homogenizing fresh tissue. Lipolysis rates in Journal of Dairy Science Vol. 74, No. 2, 1991

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isolated adipocytes are much closer to those estimated from HSL activity (36, 45, 62, 83, 84) than to those in intact tissue. An important unknown in lactation and in adipose metabolism in general is the relationship of hormone binding to CAMPsynthesis to lipolysis (39). Sidhu and Emery (59) demonstrated an in vitro lipolytic response to CAMP in adipose homogenates from lactating and nonlactating cows. This was determined as a release of FFA from endogenous triglycerides in the homogenates, not ftom added radiolabeled substrate. They were unable to determine a significant increase in HSL activity in five lactating versus four nonlactating animals. However, results from recent work involving animals sampled throughout lactation demonstrate that an increase in in vitro HSL can be detected and is at least qualitatively consistent with in vitro and in vivo lipid flux. However, the lack of closer correlation between HSL and in vitro flux remains bothersome. In tissue incubated for 15 min or 2 h (under the same conditions of in vitro glycerol release incubations) and immediately homogenized and assayed, no measurable increase was found in HSL related to unincubated tissue. There also was no increase in response to CAMP and ATP additions to the homogenates, and additions of protease inhibitors did not change the results (Sun and McNamara, unpublished observations). Thus, assay of tissue cytoplasm under saturating substrate conditions can detect gross differences in HSL activity but perhaps lacks the sensitivity to detect differences in activated (phosphorylated) or basal (nonphosphorylated) HSL. In addition, such assays appear to lack the sensitivity to physiological state and end+ crine regulators possessed by tissue slices or isolated adipocytes. This is not surprising, because the increase in HSL phosphorylation and activity occurs much faster than 15 min and dephosphorylationis continuous (11,12). Thus, a more specific assay of actual HSL activity (maximum phosphorylated versus basal) will require a tissue or cell system that maintains the structural integrity of the enzyme environment and sensitivity to B-adrenergic stimulation and in which reesterification is minimal. Such a system has been reported for human adipocytes (11, 12). J o d of Dairy Science Vol. 74, No. 2, 1991

Adipose Metabolism Differences Due to Genetics, Dietary Energy, and Level of Production

A brief qualitative summary of the genetic and dietary effects on adipose metabolism is given in Table 1. In first lactation animals, lipogenesis and esterification were lower in HG animals and in animals fed low energy rations; fastest rates were in LG animals fed high energy (HE) rations (termed LH animals). Slowest rates were seen in HG animals fed LE rations (temed HL animals) (43). This relationship held from 15 through 60 d postpartum, but by 180 d all groups had similar high rates of anabolism. Lipogenesis and esterification were most closely related (using multiple regression) to net energy intake and body weight (45). These rates were negatively related to milk energy secretion, but this explained much less of the variation than did energy intake (45). Lipogenesis, esterification, and LPL were exponentially related to energy balance at 60 & the increase in lipogenesis began at approximately 0 energy balance (45). These findings suggest that selection for milk production and lower energy intake both decrease adipose tissue anabolism in early lactation; the greatest effector is energy intake. Similar relationships exist between adipose lipid metabolism and whole animal energy status in goats. In lactating goats, LPL and acetyl coenzyme A (CoA) carboxylase activities are exponentially, positively related to net energy balance (20). The study demonstrated a set of biologically meaningful relationships among energy balance, adipose LPL, acetyl CoA carboxylase and glucose-6-phosphate dehydrogenase, long-chain fatty acids in milk, and plasma FFA. 'Ihese empirical relationships may be of limited use in predicting performance, but they point out 1) specific pathways that are most closely related to production and thus most amenable to regulation and manipulation, and 2) that in vitro metabolic rates can and do relate in a biologically consistent manner to whole animal energy utilizations. production and energy balance also have effects on in vitro basal and stimulated lipolysis in first lactation heifers (44,45) and on FFA and glycerol kinetics in cows and goats (10,23, 26). Lipolysis rates, basal or stimulated, and HSL activity (per gram of tissue, per cell or per milligram of protein) are highest in HG animals

w)

SYMPOSIUM: NONMAMMARY METABOLISM

and in LE fed heifers during the period 30 to 180 d (44,46, 62). The in vitro mean differences among genetic lines in glycerol release and HSL are of the magnitude 10 to 20% (43, 62). which agrees with the mean differences in milk production (44, 62). There is also an environment-genotype interaction in HSL expression in adipose tissue of lactating animals. Although HSL is higher (R.05)in HG than in LG animals (corrected for diet) in early lactation (30 to 60 d), most of this increase is accounted for by HL animals (45, 62). Lipolysis also relates closely to milk energy secretion and maintenance requirements as a function of BW (44, 45). Over 50% of the variation in in vitro stimulated lipolysis was accounted for by milk energy secretion, BW, and net energy intake at 60 d of first lactation (45). The majority of this was variation due to milk energy secretion. Stimulated lipolysis also related negatively, exponentially, to net energy balance (49, which accounted for 19% of the variation in this metabolic pathway at 60 d of lactation. Similar results have been obtained in lactating goats (23,26) and multiparous cows (9, 10, 68). Using radiolabeled FFA to monitor FFA flux in vivo, a close negative correlation was found between nonestefied fatty acid entry rate and energy balance, both in lactating goats and in cows in which a range of milk production and net energy balance were produced with bovine somatotropin (9, 10, 68). These in vivo results are consistent with the in vitro results and also include the contribution of total adipose tissue mass to whole animal lipid flux. Thus, recent work has further defined the understanding of adipose tissue metabolism. High milk production animals have qualitatively and quantitatively different characteristics of adipose metabolism than do lower producing animals. Although the largest differences in metabolism are expressed for HL animals, not all of the differences are due to changes in energy balance. A combination of in vivo and in vitro experiments are needed to develop further refined knowledge on the mechanistic regulation of lipid metabolism in lactation. For 50 yr, energy efficiency of milk produo tion has been thought to have a relatively high phenotypic and genotypic comlation with milk yield (13). Recent experiments are consistent with this concept (17), which is not surprising,

IN SUPPORT OF LACTATION AND GROWTH

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because increased milk yield dilutes maintenance and thus increases gross efficiency. However, a mixture of nutrients more exactly meeting the mammary gland requirements may increase the partial efficiency of milk production (2,7,8). This nutrient mix is determined in part by the metabolism in the adipose tissue, and animals of greater merit for milk p'oduction probably also have adipose tissue that is better suited to supply the correct amounts of fatty acids. Whether this difference is inherent in the tissue or is a result of different endocrine or neurocrine signals in animals differing in genetic potential remains to be determined. Adlpose Tissue Cellularity Durlng Lactation

The metabolic adaptations described in this study are based largely on metabolic rates per unit of tissue weight. This is a valid and biologically meaningful method for describing metabolism, especially when the rates are being related to the physioIogical state and metabolic performance of the whole animal. However, it cannot fully describe the cellular and subcellular mechanisms involved in adaptations to physiological state. Adipocyte size decreases in early lactation and then increases as body lipid is replenished (52, 61, 62, 73); therefore, a description of adipose tissue cellularity during bovine lactation was needed prior to more detailed interpretation of the metabolic adaptations. Lipolysis rates increased during lactation in sheep, whether rates were expressed on a tissue or cellular basis (73, 74, 76, 77). Data collected from studies using isolated adipocytes have been consistent with those using tissue slice incubations and whole animal preparations. Little or no information has been available on specific changes in fat cell size or density per unit of tissue during bovine lactation, and there has been no information on the effects of genetic selection and dietary energy throughout a lactation. Such information would not change the fact that adipose tissue adapts at the cellular level to lactation state. Thus, arguments about the proper way to express adipose tissue metabolism rates are at best irrelevant and at worst counterproductive. The ultimate goal is to predict performance given a set of environmental and genetic inputs, and a description of adipose cellularity is unnecessary for meeting that goal. Journal of Dairy Science Vol. 74, No. 2. 1991

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Cellularity descriptions may be useful for describing the types of adipose anatomy that best support the lactational state (51, 52). Such information may point out a difference in the type (size) of fat cell that is most or least responsive to the endocrine signals of a specific physiological state. This information could be used to select for animals best able to support the energy needs of lactation. In addition, fat cell size accounts for the large majority of changes in body fatness in mature ruminants (51, 53). Thus, knowing the changes in adipocyte size can allow inferences about and perhaps prediction of changes in body fatness. Multiparous bovine animals had a reduction in fat cell diameter of both omental and subcutaneous depots during early lactation (61). This was approximately 20% in omental and 30% in subcutaneous depots (61) or a calculated reduction in volume of 50 and 63%. These animals were in negative energy balance throughout this period. In Fist lactation heifers, we determined a reduction in fat cell volume of 15%from 30 d prior to and 60 d after parturition (62). However, in HH and HL animals this was approximately 35% and in LH and U animals only 10%(LG animals fed low energy rations). In HL animals, which were in negative energy balance, the reduction in adipoqte volume was 46%.However, in HH animals, adipocyte volume was reduced 30%, even though the animals were near or above energy balance. Thus, bovine subcutaneous adipocyte volume is reduced, even in first lactation heifers, and the reduction in size is sensitive to genetically d e termined milk production as well as to energy balance. Fat cells returned to their prepartum size by 120 d in LH and LL animals but remained much reduced in HH animals until 180 d. In HL animals, prepartum fat cell size had not been restored by 240 d, even though they were on ad libitum feed from 150 d. These cellularity data were consistent with the milk fat output and energy intake of these animals, such that the animals with the highest requirement for fat and the lowest intake mobilized the most fat and took the longest time to restore it (62). Marked differences were also noted in the distribution of fat cell sizes (62). More than threequarters of the adipose lipid was stored in the largest onequarter of fat cells (diameter greater than 140 pm). This population of fat Journal of Dairy Science Vol. 74, No. 2, 1991

cells was most sensitive to lactational state, energy intake, and milk production. Cells greater than 140 pn in diameter accounted for more than half of the total lipid volume lost in early lactation. In the HL animals, the lipid contained in this sue range of cells was not restored to prepartum values by 240 d. This indicates that milk production and energy intake affect fat cell size populations differently during lactation. Whether this is relevant to the fat economy of the animal has yet to be determined. The adipose cellularity data are also consistent with our understanding of adipose and lipid metabolism in lactating dairy cattle. The prepamm and immediate postpartum adaptations in lipid flux result in smaller adipocytes by 30 d postpartum, and this size reduction is sensitive to the net rate of mobilization. The nadir of fat cell size is a function of both milk fat output and dietary energy input. The rate of fat cell size restoration is also dependent upon the continuing positive fat balance of the animal. Fa&cell size in one depot is not perfectly correlated with body fatness. However, in mature ruminants, changes in fat cell size account for the large majority of changes in body fatness (51, 52, 53). Equations relating BW and mean fat cell diameter to amount of body fat in growing and mature Holsteins have been proposed by Robelin et al. (53). Such a technique could prove especially useful in lactating animals, because the determination of body water space and the calculation of body fat is costly and highly variable (14, 24, 25, 53). These equations have not yet been validated in lactating Holsteins; however, they may be used initiaUy to demonstrate changes in body fatness. From the BW and fat cell size data from our heifers (62) and equations of Robelin et al. (53) for Holsteins of similar weight, an average of 80 f 3.9 kg of lipid in 620 f 12 kg (n = 14) heifers at -30 d was obtained. This decreased to 51 f 3.5 and 527 f 9.7 at 30 d. Even considering that the calculated prepartum body lipid value must be inflated from inclusion of the conceptus in the BW, these calculated values are consistent with the small amount of information available on actual fat loss in early lactation (2, 5, 6, 7, 81, 82). Results consistent with the genetic and dietary treatments were also obtain&, all animals had about 50 kg of

SYMPOSIUM NONMAMMARY METABOLISM IN SUPPORT OF LACTATION AND GROWTH

lipid at 30 d; but LH animals restored lipid to 84 kg by 120 d and LL animals by 240 d. Meanwhile, HH animals remained below 75 kg of lipid to 180 d, and HL animals stayed below 67 kg of lipid until 180 d but restored to 87 kg of lipid at 240 d. These speculative calculations have not been validated against carcass composition but present the hypothesis that body lipid can be calculated from BW and specific fat cell parameters. The technique does not remove all the problems associated with variation in BW and body water, but it does provide consistent preliminary estimates and has the promise of being useful in studying body fat metabolism in la0 tating animals. Integrating Adipose Metabolism Into Mechanistic Models to Predict Animal Pertormance

Most information on metabolism during lactation of dairy cattle (and other species) has been qualitative: describing mean differences due to physiological state or treatment group mean differences. More recently, tissue metabolism has been related to whole animal inputs and outputs using regression techniques. These latter studies more specifically point out important metabolic pathways, but those studies are still empirical in nature. To be truly predictive, data sets must be collected that parameterize pathways: definitions of substrate concentraof the tions, K, for the substrates, and V, reaction. In this way, if inputs (dietary substrates, uptake at the intestine) are known, then animal metabolism may be estimated mechanistically, and production can be predicted (1, 2, 4, 5). Knowing these Parameters for the key pathways also allows for formdating specific hypotheses concerning metabolism (1, 2, 3, 5, 6, 49, 54) for further experimentation. For example, the specific effect of certain hormones or pharmacological agents or the presence, absence, or expression of a certain enzyme could be determined along with the effect on the overall performance of the animal. Recently, Baldwin et al. (1.2, 5) proposed a working mechanistic model of dairy cow metabolism. The equations in this model are predicated on normal Michaelis-Menten behavior of most pathways, provisions for those pathways that do not strictly follow such behavior, and the hown substrate interrelationships common

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to the key pathways. In addition, the model is constructed such that it is dependent upon the concentrations of key nutrients and metabolites and based on a moles per day quantitification of specific products (palmitate, glucose, etc). These attributes make exact knowledge of metabolism in all the various tissues involved irrelevant, at least as model behavior is presently concerned, but thus require that knowledge of whole body flux is available or estimable. Provisions are also in place for overall changes in metabolism due to physiological state (e.g., the presence of a “lactation horof milk mone” will alter the K, and,,V synthesis and maximum lipolysis in adipose tissue). The simplifying assumptions involved in the model are covered elsewhere (1, 2, 5); however, this proposed model is doubtless the most complete summary of knowledge available for dairy cattle metabolism. It provides a reference point to challenge and improve with data on metabolic kinetics from lactating dairy cattle in various physiological and endocrine states. Recently collected data can be used to estimate V,, parameters of adipose tissue lipogenesis and esterification. The V, values for these pathways were calculated on a moles per day basis from in vitro tissue metabolism and calculated body lipid during various stages of values prolactation (43, 44, 62). The,,V posed in the model were consistently higher (by 20 to 80%) than those estimated from our data. of acetate conversion to For example, the,V triglyceride storage is proposed as 37.3 moVd. Using our lipogenesis rates determined at saturating acetate and glucose concentrations and assuming 50 kg of body lipid, our estimates ranged from .03 moVd in HL animals at 15 d to 30 moUd in LH animals at 120 d. Thus, in energy-restricted animals, the actual,,V for acetate conversion to body lipid is very small, but in animals in positive energy balance the observed in vitro maximum capacity approaches the theoretical maximum. The V,, in the model represents the potential maximum in any physiological state, whereas those derived in specific from our heifers estimate the V, animals in specific states. The origin of the model parameters was primarily h m data obtained in isolated a d i p y t e s from animals very similar to the LH ones in our study (2, 6, 83, 84). Thus, the proposed,,V estimates in the Journal of Dairy Science Vol. 74, No. 2, 1991

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model appear to be consistent and biologically sensible. By using studies designed to estimate body fat and rates of metabolism generated under Michaelis-Menten conditions, it should be possible to challenge model behavior in various lactational states (e.g., amount of body fat, dietary energy input, milkfat output, concentration of “endocrine regulatod’). In addition, the model behavior in high producing, low energy balance animals Could be tested Specific kinetic data are still lacking on the major pathways of metabolism in dairy cattle, especially on the effects of endocrine and neural signals. Testing of this and other models should provide opportunities for useful research for many years to come. Regulation of Adipose Metabolism During Lactation

A varie$y of mechanisms exist for the coordinated changes in adipose metabolism during lactation; excellent reviews are available (7,8, 27,35,77).Only a brief description inmrporating the newest information on possible mechanisms regulating adipose tissue metabolism will be provided. The anabolic and catabolic pathways are regulated variously by circulating concentrations of their substrates, ions, insulin, glucagon, E, NE,somatotropin,prolactin, e s m gen, progesterone, glucocorticoids, thyroid hormones, and also possibly adenosine (71,74,77, 79). Lactation is most closely associated with changes in the concentrations of insulin, somatotropin, steroids, prolactin, and the various substrates. In most cases, one or more of the signals affect both anabolic and catabolic paths (e.g., insulin will decrease CAMP, resulting in decreased lipolysis with a simultaneously increased glucose flux,increasing lipid synthesis, and eventually causing increased protein synthesis for enzymes catalyzing most anabolic reactions). The endocrine changes that most specifically relate to the metabolic shifts in adipose tissue in early lactation are the decrease in insulin, the increase in somatotropin, and the virtual a b sence of progesterone (7,8,35).The qualitative adaptations have been used to propose mechanisms for metabolic regulation in lactation (7, 8), but sufficiently detailed data is still lacking on the direct effects of these regulators on metabolism in adipocytes in various pregnancy, J o d of Dairy Science Vol. 74, No. 2, 1991

lactational, and environmental states. Further, it is now mderstcd that one signal may increase or decrease the receptor numbers or binding capabilities for another signal (7, 8, 39, 79), thus incming the redundancy and complexity of the regulation. Adaptations in receptors that are pertinent to adipose tissue of lactating animals include decreases in insulin receptor numbers, probably due to changes in progesterone, prolactin, or both (7, 8, 15, 16, 37, 38, 63, 64, 65, 77); increases in fLadrenergic receptors and responsiveness (7,8, 32, 33, 41, 42,44,46, 48, 69, 73); and increased responsiveness to adenosine, which inhibits badrenergic action (71,74,75, 76). All of these possible changes in receptor characteristics result in changes in intracellular CAMP,either acutely or chronically. In addition to the acute effects on metabolism, the chronic effects include altered synthesis and degradation of specific enzymes. Bovine adipose tissue possesses responsiveness to a-adrenergic agonists as well (19,60); however, a decreased sensitivity to alpha stimulation, which would increase lipolysis, does not appear to be involved in the lactational adaptation of lipolysis in the bovine (19,60). Although many of the lactational adaptations in adipose tissue can be explained by these shifts in hornone receptors, direct experimental descriptions of the mechanisms involved at the adipose tissue or adipccyte are limited. Much of this lack is due to the difficulty in culturing isolated adipose tissue or adipocytes for periods of time sufficient to discern such effects. Recent progress is discussed by Vernon (79). Nevertheless, somatotropin, progesterone, estrogen, and prolactin very likely can result in an alteration in insulin or adrenergic receptors at the a d i p y t e (7,70,75,76).Mammalian metabolic regulation is often redundant, and just because an effect may be proven in isolation in vitro does not mean that the effect is of the same magnitude in vivo. The question of whether changes in numbers or binding kinetics of hormone receptors or postreceptor adaptations play a major role in adipose metabolism during lactation is a critical one. The amount of endocrine or neurocrine signal, the receiver for the signal, the intracellular messenger from the receiver, and the ability of the cell to respond to the messenger are all important to the eventual net uptake, utilization,

SYMPOSIUM: NONMAMMARY METABOLISM IN SUPPORT OF LACTATION AND GROWTH

and output of metabolites (39). Postreceptor adaptation means that the internal structure of the cell has changed to support one set of pathways over another. Such an adaptation originally may have been due to a change in substrate concentration, hormone concentration, or receptor expression or behavior. In the lactational state, insulin receptor numbers decrease in adipocytea of many species, and the reduction in anabolic and increase in catabolic pathways can be explained in large part by such a phenomenon. However, other researchers have found a reduced response to insulin in adipocytes in pregnancy and lactation and have interpreted this as a postreceptor phenomenon; that is, the response to the same amount of signal is attenuated. Likewise, the increases in,,V of lipolysis to in vitro and in vivo stimulation have been interpreted as being posueceptor adaptations (40,55,56). However, in vitro data also suggest that the p-adrenergic receptor pop ulations are altered due to lactation or somatotropin (79). These are not conflicting, but rather, incomplete results with conflicting interpretations. Changes in receptor numbers, affinity, second messenger coupling, and enzyme response are not mutually exclusive but represent a coordinated adaptation to a new state. This confusion stems from our limited understanding of the mechanisms linking signal to receptor to messenger to biological response, especially in any quantitative sense (39). Whole animal and tissue response studies can suggest, not prove, that a change occurs in only receptor or only postreceptor mechanisms. The biological principles, as we understand them, and available data suggest that the recep tor and postreceptor adaptations are intrinsically linked such that a change in one leads to a change in the other. For example, a decrease in insulin receptors may be a direct or indirect cause of an increase in padrenergic receptors (or vice versa). After a cell has adapted to a new physiological state, the intracellular structure is physically altered, and the response of a cell to a stimulus or substrate also is altered because the old structure no longer exists. Thus, attempting to define receptor and postreceptor-mediated adaptations without thorough studies at eveIy level of organization becomes another of the many futile cycles of research activity. An exciting recent finding is that the whole body is more responsive to an E challenge after

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chronic treatment with somatotropin (40, 56). This response, measured as an increase in FFA for a period of time following an acute dose of E, was related to the chronic dose of bST used and the resultant milk production (56). The response appeared to be due more to an increase in maximum response than to sensitivity. This result is consistent with and extends the original work of Sidhu and Emery (58, 59) on increased whole body response to E challenge in early lactation and is consistent with the in vitro increase in lipolysis,,V in lactation and in relation to production (45). The result obtained in bST-treated animals is further evidence that, regardless of the reason for increased mammary activity, the lipolytic ability of the adipose tissue relates directly to activity of the mammary gland. The causality and mechanisms of this relationship are not yet understood. Although summary figures are usually oversimplifications of the facts, Figure 1 is an attempt to describe briefly the effects of lactation, genetic selection for xnilk production, and dietary energy on the intracellular regulation of lipolysis in dairy cattle. Lipolysis is directly and chronically regulated by the release of NE from the SNS (28). and this regulation is involved in the adaptations of lipolysis in various physiological and nutritional states (34). In rodents, activity of the SNS, measured by NE turnover, is altered during pregnancy and lactation to conserve energy utilization in brown adipose tissue (66, 67, 80). However, in dairy cows, &adrenergic blockade did not alter the nocturnal increase in FFA (30), which argues against the involvement of the SNS in this phenomenon. Unfortunately, that study (30) was not designed to determine SNS activity in lactating versus ncmlactating animals, and there were no data presented for untreated control animals. Dairy cows did increase their whole body response to infusions of E after 24 to 72 h of fast; the greatest response was aftm the longest fast.. Whether or not altered SNS activity is a causative mechanism in the increased lipolysis during lactation is still hypothetical. However, keeping in mind the relationship between mammary activity and adipose lipolysis, it is possible that the SNS system is a critical link in communication of the mammary gland and adipose tissue during lactation. J o d of Dairy Science Vol. 74, No. 2, 1991

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SNS ( ? )

I NE

I

Adrenergic receptor ( L t ) \

9

Adenyla e c y c l a s e ( L ? )

L

CAMP (L-;G?)

/ J Adenosine ( L , t ;G? )

Protein kinase ' A ' ( ? ) Hormone s e n s i t i v e l i p a s e (L,t; G,t; E , t ) \

4 L i p o l y s i s (L,t;Gt;Et)

Figure 1. Changes in inhacellular regulation of adipose tissue lipolysis due to lactation, genetic selection, and dietary energy. The graph represents the major flow of regulation from fbadrenergic receptor binding of catecholamines to the biological response, release of fiee fatty acids and glycerol. The notations in parentheses are as follows: L is the effect of the lactational state in itself, in this case primarily eariy lactation; G is the effect of genetic selection for increased milk production, such that an increase in the variable was seen in animals of higher merit; E is the effeet of diebuy energy; in this case, a reduction in dietary eaergy intake caused an increase in the variable. A question mark notes where no clear information is available, a dash indicates no effect. SNS = Sympathetic nervous system; NE = norepinepm; CAMP = cyclic AMP.

CONCLUSIONS

Hypotheses and Challenges for Future Research

With the information available on animal, organ, cell, and subcellular and molecular adap tations to lactation by adipose tissue, several hypotheses can be proposed. What is needed are studies designed to determine kinetic parameters for adipose metabolism pathways at different lactation stages and milk yields, fat and energy intake, and energy balance. Work in this area should include estimation of the K, and,,V of these pathways in early versus other lactational states because of changes in energy balance and milk energy secretion and because of genetic selection, somatotropin treatment, and feeding of high fat rations. Simple determination of single point metabolic rates (which is common with in vitro tissue Journal of Dairy Science Vol. 74, No. 2, 1991

incubations) can easily miss the real effects of such treatments. The effect on carbon flux through the pathways over a range of substrate and inhibitor concentrations must be known in order to determine the true effects on intermediary metabolism. Two other areas that need work are the role of steroids in regulating adipose metabolism in lactation and pregnancy and the effect of delivery of fat directly to the small intestine on adipose metabolism during lactation. Another needed area of study is the definition of specific genetic differences in enzyme and mRNA expression and turnover and the resultant effects on metabolite flux and kinetic parameters in adipose tissue (e.g., the question of whether there are more copies of HSL -A, structural genes, or transcription initiators in high producing animals or whether mRNA turnover is altered). The possible adaptations in insulin release, insulin-like growth factors, SNS activity, and the receptors for these signals must be investigated to complete the picture of metabolic regulation of adipytes. Changes in receptor numbers or binding should be related to changes in biological response (39) to improve our ability to predict the effect of a change in endocrine environment. The effect of body fat content at calving on subsequent adipose metabolism and overall energetic efficiency during lactation will be useful in making recommendations to our producers on the management of the total animal during the lactational cycle (31, 81, 82). New evidence suggests that higher reserves of body fat at parturition increase performance and energetic efficiency if managed correctly (31). However, the organ and cell level determinants of such a possible result are unknown. In this area, easier, more direct ways of estimating body fat (e.g., by using fat cell size) may be useful. Because of the central role of lactation in nurturing the young of all species and because of the special role of the lactating ruminant in converting unpalatable and fibrous plant material into human food, research on the lactational state is a necessity. Because of the role of adipose tissue in buffering the mammary gland from the external environment and providing the optimum mix of metabolites, more specific research is needed The adaptations of adipose tissue to lactation provide an outstanding model

SYMPOSEUM: NONMAMMARY METABOLISM IN SUPPORT OF LACTATION AND GROWTH

of the molecular adaptations to changes in nutrient influx and endocrine environment. Also, the adipose adaptations to lactation offer an excellent opportunity to provide quantitative data relating, in a predictive manner, the metabolism in one organ to the effect on the metabolism and production of the whole animal. REFERENCES 1 Baldwin, R. L., J. France, D. E. Beever, M. Gill, and J.H.M. Thomley. 1987. Metabolism of the lactating cow. III.Ropesties of mechanistic models suitable for evaluation of energetic relation&$s and factors involved in the partition of nubients. J. Dairy Res. 5 4 133. 2 BaldR L., J. Fran~e,and M. Gill.1987. Metabolism of the lactating cow. I. Animal elements of a mechanistic model. J. Dairy Res. 5477. 3 Baldwin. R L., N. E. Forsberg, and C. Y. Yu. 1985. Potential for altering energy partition in the lactating cow. J. Dairy Sci. 68:3394. 4Baldwin, R. L., and N. E. Smith. 1971. Intermediary aspects and tissue interactions of ruminant fat metabolism. J. Dairy Sci. 54583. 5 Baldwin, R L., J.H.M. Thomley, and D. E. Beever. 1987. Metabolism of the lactating cow. DI. Digestive elements of a mechanistic model. J. Dairy Res. 54107. 6 Baldwin, R.L., Y.T. Yaog, K. Crist, and G. Grichting. 1976. Theoretical model of ruminant adipose tissue metabolism in relation to the whole animal. Fed. Pmc. 35:2314. 7 Bauman, D. E., and W.B. crurie. 1980. Partitioning of nutrients during pregnancy and lactation: a review of mechanisms involving homeostasis and hommrhesis. J. Dairy Sci. 63:1514. BBauman, D. E., and I. U Elliot. 1983. Control of nutrient partitioning in lactating ruminants.Page 437 in Biology of lactation. T. B. Mephatn, ed. Elsevier Publ. BV, Amsterdam, Neth. 9 Bauman, D. E., P. J. Eppa~d,M. J. DeGeeter, and G. M. Lanza. 1985. Responses of high producing dairy cows to long term treament with pituitary somatotropin and recombinant somatotropin. J. Dairy Sci. 68: 1352. lOBauman, D. E., C. J. Peel, W. D. Steinhour. P. J. Reynolds, H. E Tyrrell, A.C.G. Brown, and G. L. Haaland. 1988. Effect of bovine somatotropin on metabolism of lactating dairy cows:influence on rates of irreversible loss and oxidation of glucose and nonesterified fatty acids. J. Nub. 118:1013. 11Belfrage, P., G. Fredrichn, H. Olsson, and P. Stralfors. 1983. Control of adipose tissue lipolysis by phosphorylation/dephosphorylation of hormone-sensitive lipase. The adipocyte and obesity: cellular and molecular mechanisms. A. Angel, C. H. Hollenberg. and D.A.K. Roncari, ed. Raven Press, New Yo& NY. 12 Belfrage, P., G. Fredrickson, H. Olsson. and P. Stralfors. 1984. Regulation of adipose tissue lipolysis through revmible phosphorylation of hormone-sensitive lipase. Adv. Cyclic Nucleotide Protein Phosphorylation Res. 17:351.

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on lipogenesis in rat adipose, liver and Imamnary tissues. J. Nu@. 108514. 3OK)Frohli, D. M.,and J. W. BhmL 1988. Nonestedied fatly acids and glucose in lactating dairy cows: diarnal variations and changes in responsiveness during fasting to epinephrine and effects of bcta-adremrgicblockade. J. Dairy Sci. 71:1170. 31 Gamsworthy, P.C. 1988. The effect of energy reserves at calving on performance of dairy cows. Page 151 in N~riti~ and n lactation in the dairy COW. P. C. Gamsworthy. cd, Butterworths, Boston, MA. 32Guesnet, P., M. Massoud, and Y. Demame. 1987. Efem of pregnancy and lactation on lipolysis of ewe adipocym induced by padrmeqic stimnlatioe Mol. Cell. Endocrinol. 50177. 33 Jasta, E.H., and T.N. We-. 1980. Beta-adrenergic receptor involvement in lipolysis of dairy cattle sub cutarmus adipose tissue during dry and lactating state. J. Dairy Sci. W1655. 34 Johnston. J. L. 1987. A role for norepinephrine metabolism io energy balance and obesity: a review. Can. Inst. Food Sci. Tfeclmol. J. Xk331. 35 Kallchoff, R. K., A. H. Kissebah, and H. J. Kim. 1978. Carbohydrate and lipid metabolism during normal pregnancy: relationship to gestational hormone action. semie Perinatal. (NY) 2291. 36w00, J. C., M. Yamamoto, M. Kawamura, and D. Steinberg. 1983. Hormone-sensitive lipase system and insalin stimulation of protein phospaetase activities in 3T3-Ll adipocytes. Page 225 in The adipocyte and obesity: cell and molecular mechanisms. A. Angel, C. H. Hollmberg, and D.A.K. Romui, ed. Raven Press. New Yo&, NY. 37Knhn, N. J. 1969. Progesterone withdrawal as the lactogmic trigger in the rat. J. Endocnm, . 1. w 3 9 . 38 Knhn, N. J., and J. M.Lowenstein. 1967. Lactogmesh in the rat: changes in metabolic parameters at partmition. Biochem. J. 105:955. 39 Lmb, J. N.. and S. Stricwand. 1987. Hormone binding and coupled nspoosc relationships in systems depmdent on the generation of s c a m b y medii. Mol. Cell. Endocrinol. 1:75. 40 McCutcheon, S.A., and D. E. Bamnau 1986. Effect of chronic growth honnonc treammlt on respoILEes of epinephrine and thyrotropin-releasing hormone in lactating cows. J. Dairy Sci. 6944. 41 McNamara, J. P. 1988. w o n of bovine adipose tissue metabolism during lactation 4. Dose-responqiveness of epinephrine as altered by stage of lactation. I. Dairy Sci 71643. 42McNamara, J. P., and J. K. Hillers. 1986. Adaptations in lipid metabolism of bovine adipose tissue in lactogenesis and lactation. J. Lipid Res. 27:150. 43 McNamara, J. P.. and J. K.Hillers. 1986. Regulation of bovine adipose tissue metabolism during lactation. 1. Lipid synthesis in response to both increased milk production and decreased energy intake. J. Dairy Sci. 69:3032. 44 McNamara, J. P., and J. K. Hillers. 1986. Regulation of bovine adipose tissue metabolism during lactation. 2. Lipolysis response to milk production and energy intake. J. Dairy Sci. 693042. J. P.. and J. K. Hillers. 1989. Regohtion of 45 McNbovine adipose tissue metabolism during lactation. 5. J o d of Dairy Science Vol. 74. No. 2, 1991

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