Adaptations of Glucose Metabolism During Pregnancy and Lactation

Journal of Mammary Gland Biology and Neoplasia, Vol. 2, No. 3, 1997

Adaptations of Glucose Metabolism During Pregnancy and Lactation Alan W. Bell1,2 and Dale E. Bauman1

Increased glucose requirements of the gravid uterus during late pregnancy and even greater requirements of the lactating mammary glands necessitate major adjustments in glucose production and utilization in maternal liver, adipose tissue, skeletal muscle, and other tissues. In ruminants, which at all times rely principally on hepatic gluconeogenesis for their glucose supply, hepatic glucose synthesis during late pregnancy and early lactation is increased to accommodate uterine or mammary demands even when the supply of dietary substrate is inadequate. At the same time, glucose utilization by adipose tissue and muscle is reduced. In pregnant animals, these responses are exaggerated by moderate undernutrition and are mediated by reduced tissue sensitivity and responsiveness to insulin, associated with decreased tissue expression of the insulin-responsive facilitative glucose transporter, GLUT4. Peripheral tissue responses to insulin remain severely attenuated during early lactation but recover as the animal progresses through mid lactation. Specific homeorhetic effectors of decreased insulin-mediated glucose metabolism during late pregnancy have yet to be conclusively identified. In contrast, somatotropin is almost certainly a predominant homeorhetic influence during lactation because its exogenous administration causes specific changes in glucose metabolism (and many other functions) of various nonmammary tissues which faithfully mimic normal adaptations to early lactation. KEY WORDS: Pregnancy; lactation; glucose metabolism; insulin responses; homeorhesis.

such as pregnancy toxemia or lactation ketosis. This imperative has led to the evolution of metabolic adaptations in maternal nonuterine and nonmammary tissues which are regulated and coordinated to ensure that glucose supply to the gravid uterus and lactating mammary gland is buffered against variations in maternal nutrition and other environmental influences. This review will focus on recent advances in our understanding of the cellular, tissue and whole-animal metabolic bases for enhanced glucose synthesis and attenuated glucose utilization in nonuterine and nonmammary tissues. It will particularly emphasize emerging concepts which seek to explain extracellular regulation and coordination of multiple adaptations in organs and tissues as diverse as liver, skeletal muscle, and adipose tissue. Most examples will be taken from the literature on domestic ruminants because of the wealth of in vivo data in species such as sheep, dairy

INTRODUCTION Glucose is a primary nutrient for conceptus growth and milk synthesis. Together with its glycolytic metabolite, lactate, glucose is the most important source of fuel for oxidation in fetal and placental tissues. It is also a vital oxidative and synthetic substrate for mammary metabolism, including lactose synthesis which, in most species, is the primary osmotic determinant of milk volume. Provision of glucose for uterine or mammary utilization is therefore a metabolic priority for the pregnant or lactating mammal, disruption of which can lead to serious ketoacidotic syndromes ' Department of Animal Science, Cornell University, Ithaca, New York. 2 To whom correspondence should be addressed at Department of Animal Science, 149 Morrison Hall, Cornell University, Ithaca, New York 14853-4801. e-mail: [email protected]

265 1083-3021/97/0700-0265$l2.50/0 © 1997 Plenum Publishing Corporation

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cattle and goats (1). Therefore, our review is prefaced with a brief consideration of some of the special metabolic characteristics of these species which may not be familiar to many biomedical researchers.

SPECIAL FEATURES OF GLUCOSE METABOLISM IN RUMINANTS Ruminants are obligate herbivores whose evolutionary success has, in large part, resulted from their pregastric, fermentative mode of digestion. This allows them to efficiently utilize cellulose and other fibrous feed components, and derive much of their protein requirements from digestion of rumen microbes. However, it also ensures that more readily digestible, nonstructural carbohydrates, including starches and sugars, are subjected to microbial fermentation in the reticulorumen before they can become available for amyloytic digestion and absorption in the small intestine. This means that in the recently fed as well as the postabsorptive state, ruminants must depend almost exclusively on gluconeogenesis in liver and to a lesser extent, kidneys for their tissue glucose requirements. The importance of hepatic gluconeogenesis is highlighted by the fact that glucose requirements are more than doubled in late-pregnant ewes carrying twins, and increased 4-fold in genetically superior, lactating dairy cows compared to their nonpregnant or nonlactating counterparts. In some truly exceptional cows whose average milk yield has approached 90 L/d, the requirement for mammary lactose synthesis alone must necessitate a 7-fold increase in glucose production. In well-fed ruminants, the principal precursor for hepatic gluconeogenesis is propionate, one of the major volatile fatty acid (VFA) byproducts of pregastric fermentation, which is absorbed via the ruminal epithelium into portal venous blood and almost quantitatively removed by the liver (2,3). The rate of ruminal production of propionate and other VFA is directly related to dietary intake of fermentable substrate; propionate synthesis is especially favored by fermentation of starches by amylolytic bacteria (2, 4). Since hepatic supply of propionate is a principal determinant of hepatic glucose synthesis (3), it is not surprising that in all classes of ruminants whole-body glucose production is highly correlated with digestible energy intake (2,3). As the supply of propionate dwindles, the importance of other glucogenic substrates, such as lactate, amino acids, and glycerol, increases (3). Hepatic utili-

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zation of the latter glucose precursors is more clearly influenced by the positive and negative effects of glucagon and insulin, respectively, than is that of propionate (3). This appears to be mediated indirectly by effects of insulin on peripheral tissue metabolism, as well as more direct hormonal influences on hepatic gluconeogenesis. The ruminant digestive mode also has a profound effect on the pattern of posthepatic nutrient supply and utilization. Acetate, derived directly from ruminal fermentation, and 3-hydroxybutyrate, derived mostly from hydroxylation in rumen epithelium of ruminal butyrate, are major substrates for oxidation in tissues such as kidneys, heart and skeletal muscle; notable exceptions are brain and liver (5). These fermentation byproducts are also important sources of carbon and NADPH (via the isocitrate cycle) for lipogenesis in adipose tissue and mammary gland (6). Thus, while some tissues such as brain have retained a mandatory requirement for glucose as a metabolic fuel, and all others require glucose for certain indispensable, nonoxidative functions, most ruminant tissues, including muscle and adipose tissue, have evolved the capacity to substantially substitute VFA and their derivative ketoacids for glucose as respiratory fuel or lipogenic substrate. Consequently, under normal physiological conditions the proportion of total glucose utilization that is insulin-dependent (10-30%) is somewhat lower in ruminants than in nonruminants (7, 8). Nevertheless, muscle, adipose tissue and other insulin-responsive tissues are as dependent on insulin for promotion of glucose transport and metabolism in ruminants as in nonruminants, as shown by the pathological effects of artificially induced diabetes (9). However, in ruminants these tissues are generally less responsive to the actions of insulin than in nonruminants such as humans, rats or pigs fed high carbohydrate diets (10). Recent evidence from application of the hyperinsulinemic, euglycemic clamp technique to sheep suggests that this is due more to constraint of maximal responsiveness than to reduced sensitivity to insulin (7, 8). This phenomenon is apparently postreceptor and consistent with a metabolic regulatory strategy that must continually balance the opposite influences of insulin and glucagon on hepatic gluconeogenesis against the effects of insulin on extrahepatic glucose utilization (11). In many ways, this situation is reminiscent of that facing nonruminants eating highfat diets.

Adaptations of Glucose Metabolism During Pregnancy and Lactation

PREGNANCY Conceptus Metabolism In well-fed, late-pregnant ewes carrying a single fetus, uptake of glucose by the gravid uterus accounts for 30-50% of an increased maternal glucose supply (12, 13). Although the placenta has only about 10% the mass of the fetus at this time, consumption of glucose by nonfetal conceptus tissues (mostly placenta) accounts for about two-thirds of total uterine uptake (12, 13). Thus, in addition to its vital function of maternal-fetal glucose transport, the placenta is a major contributor to the increased glucose demands on the pregnant animal. The ratio of placental to fetal glucose consumption is even greater during mid pregnancy (14), although absolute rates of uterine glucose uptake increase appreciably during the latter half of pregnancy (14, 15). Analysis of the kinetics of placental glucose transport in vivo has confirmed that in sheep and other species, this process is achieved by facilitated diffusion (16,17). We have recently shown that the predominant glucose transporter protein isoforms in sheep placenta are GLUT1 and GLUT3 and that both mRNA and protein abundance of these transporters, most notably those of GLUT3, increase from mid to late pregnancy (18). This developmental pattern appears to account for much of the 5-fold increase in glucose transport capacity of the ovine placenta in vivo over this period (19). As in nonruminant species (20, 21), we found negligible expression of the insulin-responsive isoform, GLUT4, in sheep placenta (18). This is entirely consistent with the lack of effect of insulin in vivo on uteroplacental uptake and placental transport of glucose in pregnant ewes (22, 23). Much of the glucose entering the fetal bloodstream is oxidized directly, or indirectly after conversion to lactate in fetal and trophoblastic tissues served by the fetal circulation (24). Oxidation of glucose and lactate together account for about 60% of ATP synthesis in the well-nourished, late-gestation fetal lamb (25). Under these conditions essentially all glucose utilized by the fetus is of maternal origin but in ewes that are fasted (26), severely underfed (27) or suffering chronic, insulin-induced hypoglycemia (28), uteroplacental uptake and placental transfer of glucose are substantially reduced. The fetus then relies increasingly on endogenous glucose production through activation of hepatic gluconeogenesis (29), for which the principal

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substrates are presumably amino acids. The latter is inferred from observations that fetal access to maternal amino acids is relatively unimpaired by maternal undernutrition (30) but the fractional rate of amino acid catabolism to urea is markedly increased (30, 31). This inevitably leads to reduced rates of fetal tissue protein synthesis (32) and growth (12, 30). During the more common but less well-studied state of moderate maternal undernutrition in late pregnancy, uterine uptake of glucose and fetal growth are often unimpaired despite a moderate reduction in maternal glucose production (10, 33). As discussed in the next section, uterine glucose uptake is supported by an array of metabolic adaptations in maternal tissues which are manifested as the "glucose-sparing effect of pregnancy." In addition, we believe that the conceptus itself is an active participant in this phenomenon, indirectly via the possible regulatory influence of placental hormones on maternal tissues, and more directly via upregulation of placental glucose transport capacity. This was demonstrated in twin-pregnant ewes restricted to ~60% of their predicted energy requirement for several weeks during late pregnancy. A clear effect on glucose partitioning was indicated by the development of maternal hypoglycemia and a reduced maternal-fetal glucose concentration gradient, but unchanged fetal or placental growth (34; Table I). In these animals, placental glucose transport capacity at 135 d of pregnancy, assessed in vivo by measurement of placental clearance of the nonmetabolizable analogue, 3-0-methyl glucose, was increased 50% by material undernutrition (34; Table I). This response was highly correlated with a similar increase in abundance of placental glucose transporters as judged from the in vitro binding of cytochalasin B, a competitive inhibitor of carrier-mediated glucose transport (Table I; Fig. 1). A modest but significant increase in placental abundance of GLUT3 protein was observed in underfed ewes; placental levels of GLUT3 mRNA and GLUT1 protein and mRNA were not significantly affected (R. A. Ehrhardt and A. W. Bell, unpublished).

Metabolic Adaptations in Maternal Tissues Pregnancy induces adaptive responses in maternal hepatic synthesis and peripheral tissue utilization of glucose which are exaggerated by moderate undernutrition. In sheep, much of the pregnancy-induced increase in maternal gluconeogenesis can be attributed

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Table I. Effects of Moderate Undernutrition During Late Pregnancy on Characteristics of Placental Glucose Transport in Ditocous Ewes at 135 d of Pregnancy (Means for 5 Ewes per Treatment) Variable

Control"

Underfed*

PSE'

P

Maternal plasma glucose, mg/dL Maternal-fetal plasma glucose gradient, mg/dL Placental glucose clearance, mL/min-kg placental wt Cytochalasin B binding sites (pmol/mg protein)

66.9 56.7 117 105

51.1 41.9 176 126

1.7 1.1 7.9 3.9

0.001 0.001 0.001 0.01

a b c

Fed at 100% of predicted feed requirements throughout late pregnancy. Fed at 60% of predicted feed requirements over interval of 121 to 135 d of pregnancy. Pooled standard error.

to increased energy intake, but glucose production increases during late pregnancy even when intake is restricted (35, 36). The glucose production is presumably supported by increased peripheral mobilization and hepatic uptake of endogenous substrates such as amino acids from muscle protein breakdown and glycerol from the mobilization of adipose tissue triglycerides. Interestingly, there is also corroborated evidence that pregnancy may somehow enhance the efficiency of hepatic synthesis of glucose from propionate (36, 37). This evidence is hard to reconcile with a more detailed analysis of isotopic carbon transfer studies which suggests that in nonpregnant, maintenance-fed sheep, hepatic use of propionate for gluconeogenesis is essentially complete (38). Glucose uptake by peripheral tissues such as hindlimb muscle and adipose tissue tends to decrease dur-

Fig. 1. Relation between maternal-fetal clearance of 3-0-methyl glucose (3MG) in vivo and concentration of Cytochalasin B binding sites in placental tissue from well-fed control and moderately underfed ewes at 135 d of pregnancy. From unpublished data of R.A. Ehrhardt and A.W. Bell.

ing late pregnancy (39, 40) although it is still not entirely clear how much the evidence for these responses in "well-fed" animals is confounded by variation in voluntary feed intake. In one study in which net glucose uptake by muscle was reduced by 35-40% in late-pregnant vs. nonpregnant ewes, the fractional conversion of glucose to lactate by this tissue was significantly increased (41), consistent with a report of pregnancy-induced increases in whole-body lactate production and recycling of glucose carbon through lactate in sheep (42). Effects of pregnancy on glucose metabolism by adipose tissue have not been studied in vivo, but the ability of ovine adipose tissue to synthesize glyceride-glycerol from glucose in vitro was substantially reduced in tissue from late-pregnant ewes (43). Muscle and to a lesser extent, adipose tissue, account for most of the ~20% of total glucose disposal which, in nonpregnant, nonlactating sheep, appears to be insulin-dependent (7, 8). It is therefore likely that altered responses to insulin in these tissues largely account for the development of moderate insulin resistance in various parameters of whole-body glucose utilization in late-pregnant ewes (8, 44). Analysis of dose-response relations between these variables and plasma insulin, obtained in hyperinsulinemic, euglycemic clamp studies, attributed much of the reduction in tissue response to reduced sensitivity to insulin, represented by increased ED50 values (Fig. 2; 8). According to Kahn's (45) model this implies reduced insulin receptor abundance and(or) activity in affected tissues, consistent with a reported reduction in insulin receptor number of adipocytes in pregnant versus nonpregnant ewes (43). The maternal insulin resistance which presumably mediates much of the glucose sparing effect of pregnancy is exaggerated by moderate undernutrition of ditocous ewes (60% energy requirement for 2 wk)


Fig. 2. Relations between insulin-dependent glucose utilization (IDGU) and plasma insulin concentration in nonpregnant fed, nonpregnant underfed, pregnant fed, and pregnant underfed ewes. Under basal conditions, IDGU accounted for approximately 21%, 37%, 11%, and 10% of total glucose utilization in these respective groups. Values are means for 5 ewes per group. Reproduced from the Journal of Nutrition (8).

(Fig. 2; 8). Cellular and molecular bases for this in vivo observation are unknown. However, by analogy with human clinical syndromes and animal models involving peripheral insulin resistance, they seem likely to involve impaired capacity for cellular glucose transport (46). It is therefore notable that in ditocous ewes restricted to 60% of predicted energy requirement during late pregnancy, as in our previous study (8), levels of the insulin-responsive glucose transporter, GLUT4, were significantly reduced in skeletal muscle and perirenal adipose tissue, but unchanged in subcutaneous adipose tissue (Fig. 3). The adipose tissue

Fig. 3. Relative abundance of GLUT4 protein in subcutaneous (SC) and perirenal (PR) adipose tissue, and semitendinosus (ST) muscle in well-fed control and underfed ewes during late pregnancy. Values are means ± SE for 5 ewes. Paired means with different letters are significantly different (P < 0.05).

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response resembles that in adipocytes from gestationally diabetic women (47) and diabetic rodent models (46). However, the clear reduction in muscle levels of GLUT4 contrasts with responses in diabetic women (48) and rodents (46), which were confined to more subtle abnormalities in basal and insulin-stimulated subcellular distribution of GLUT4. It is thus apparent that in moderately underfed pregnant ewes, the repartitioning of a dwindling maternal glucose supply to favor the conceptus involves coordinated, reciprocal adaptations in glucose transport capacity in placenta and maternal peripheral tissues. Central to these adaptations is the downregulation of insulin sensitivity and(or) responsiveness in maternal adipose tissue and muscle, related to decreased abundance of GLUT4. It remains to be determined whether such changes in tissue glucose transport are primary, rate-limiting adaptations, or are secondary to changes in intracellular glucose demand. Nevertheless, these adaptations in adipose tissue and muscle, together with the lack of effect of insulin and moderate upregulation of glucose transport capacity in the placenta, ensure that glucose is preferentially supplied to the conceptus.

LACTATION Lactogenesis and Periparturient Adaptations The dramatic metabolic changes in ruminant mammary and nonmammary tissues during the transition from late pregnancy to established lactation have been reviewed recently (49). Most notable among these is the more than doubling of mammary glucose uptake during the 2 d before parturition in goats, which preceded an even more substantial increase immediately after parturition (50). The authors of this study concluded that the magnitude and timing of the prepartum increase is an important index of the onset of copious milk secretion because glucose is required for lactose synthesis and lactose is the most important osmotic solute in milk. The impact of this sudden increase in glucose demand is highlighted by observations that it occurs without any increase in voluntary feed intake and its magnitude is as great as the whole-body glucose production of a nonpregnant, nonlactating goat at maintenance (51). A similar challenge exists for the high-yielding dairy cow, whose estimated mammary requirement for glucose a few days after parturition is

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270 more than 2.5 times that of the gravid uterus during late pregnancy (49). To meet this challenge, the rate of hepatic gluconeogenesis must abruptly double during the periparturient period without a concomitant increase in supply of propionate or any other glucogenic substrate of dietary origin (52, 53). Even generous prediction of these and endogenous sources of lactate and glycerol leaves a major shortfall in necessary substrate, leading to the conclusion that substantial mobilization of body protein reserves is necessary (49). It is presumably not coincidental that the estimated requirement of endogenous amino acids, about 500 g/d, is consistent with the magnitude of negative nitrogen balance in dairy cows during the week after parturition (54). The most likely source of mobilized amino acids is skeletal muscle. Net protein loss from this tissue is indicated by a 25% reduction in muscle fiber diameter in cows immediately after parturition (55) and a decline in muscle protein:DNA ratio during early lactation in ewes (56). These observations are consistent with the reduction in muscle protein synthesis observed in goats that were in negative nitrogen balance during early lactation (57, 58). By analogy with underfed Holstein steers in negative nitrogen balance, it might be predicted that net release of amino acids from skeletal muscle is achieved entirely by suppression of protein synthesis rather than enhancement of protein degradation (59). However, increased muscle protein degradation is strongly implied by the pronounced increase in plasma concentrations of 3-methylhistidine we and others have observed in dairy cows during the early postpartum period (Fig. 4; 60).

Fig. 4. Plasma concentration of 3-methylhistidine (3MH) before and after parturition in mature Holstein cows. Values are means with standard error bars for 10 cows. From unpublished data of W.S. Burhans, J.A. Rathmacher, and A.W. Bell.

Use of glucose for adipose tissue lipogenesis, which is already low during late pregnancy, is further suppressed to minimal levels after the onset of lactation in ewes (43) and cows (61) and is almost totally unresponsive to insulin at this time (62). The lack of insulin response could not be attributed to any change in insulin binding by adipocytes (43), implying a postreceptor phenomenon (8). Preliminary evidence suggests impairment of insulin signal transduction downstream of phosphoinositol 3-kinase (PI3 kinase) (63). Glucose utilization in skeletal muscle and other peripheral tissues apart from adipose tissue has not been examined in periparturient ruminants. However, the marked reduction in whole-body glucose oxidation in newly parturient dairy cows (53) strongly suggests reduced glucose use by such tissues, mediated by very low plasma insulin levels (64) and possibly, tissue refractoriness to insulin. As described in a later section, these nonmammary metabolic adaptations persist through early lactation until dietary intake and ruminal synthesis of glucogenic substrates comes into equilibrium with mammary glucose demands.

Mammary Metabolism The mammary glands of high-yielding dairy ruminants require enormous amounts of glucose, accounting for as much as 80% of a substantially increased whole-body supply in superior cows at peak lactation (53). It appears that in lactating cows as in nonruminants, mammary glucose transport is largely, if not solely accounted for by the GLUT1 transporter (65). As in the placenta, minimal abundance of the insulin-responsive GLUT4 isoform is consistent with the lack of effect of insulin on mammary glucose uptake in lactating goats (66) and cows (67). Interestingly, observations of a similar lack of GLUT4 expression in rat mammary tissue has led to a reappraisal of the conventional wisdom that mammary glucose uptake is regulated by insulin in this species (68, 69). It is further notable that mammary glucose utilization in lactating women is unresponsive to upper physiological elevations of plasma insulin, as demonstrated in glucose clamp studies (70). Mammary glucose uptake is also relatively independent of arterial glycemia (71), presumably because it is regulated mostly by intramammary demand. This is analogous to glucose transport characteristics of the brain which, like the


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mammary glands, relies mostly on the high capacity GLUT1 transporter for access to blood glucose (72). Intramammary metabolic disposal of glucose is dominated by lactose synthesis in most species, including ruminants, metabolic details of which are reviewed elsewhere (73). Thus, in a detailed in vivo study of mammary glucose utilization in goats, of the 85% of [U -- I4C] glucose taken up which could be accounted for, 75% appeared in milk lactose (74). Other significant metabolic fates included glyceride glycerol synthesis (4.5% oxidation to CO2 phosphate pathway). Pentose cycle activity was estimated to account for about one third of the NADPH required for mammary de novo fatty acid synthesis (74). This estimate complements the calculations of Bauinan and Davis (6) that in ruminants, most or all of the remaining NADPH is generated by the isocitrate dehydrogenase pathway.

Nonmammary Adaptations The adaptive changes in hepatic gluconeogenesis and peripheral glucose utilization which are initiated in late pregnancy and strongly reinforced at the onset of lactation were discussed in previous sections. In general, these adaptations persist through early lactation but wane in intensity as the animal's nutrient balance responds to increasing voluntary feed intake which peaks some weeks after maximal milk yield. Changes in the pattern of hepatic utilization of propionate versus endogenous glucogenic substrates have not been systematically studied across the lactation cycle. However, it can be confidently inferred from the in vivo response of hepatic gluconeogenesis to differing rates of nutrient supply in lactating cows (75) that as the dietary intake of fermentable carbohydrate increases, so too will the contribution of propionate to hepatic glucose synthesis. The greater importance of endogenous substrates such as lactate, glucogenic amino acids, and glycerol during early lactation is consistent with a greater inhibitory effect of insulin on whole-body glucose production in goats during early versus mid lactation (76) because hepatic uptake and utilization of propionate is much less responsive to insulin than that of the endogenous substrates (77). Although this greater response during early lactation occurred within the upper physiological range of insulinemia, its significance is doubtful because pancreatic responsiveness to insulinotropic agents and plasma insulin levels are greatly depressed during early lactation (64, 78).

Fig. 5. Relations between glucose utilization rate above basal and plasma insulin concentration in goats during early lactation, mid lactation, and dry (nonlactating) period. Values are means ± SE for 4 animals. *P < 0.05 compared with corresponding value during dry period; + P < 0.05 compared with corresponding value during mid lactation. Reproduced from (76) with permission of the American Physiological Society.

In contrast, the insulin responsiveness of wholebody glucose utilization was markedly less in early than in later lactating or nonlactating goats (Fig. 5; 76). As discussed earlier, some of this early insulin resistance must reside in adipose tissue (62), the lipogenic ability of which rebounds dramatically during mid lactation (61). This enhanced capacity for de novo fatty acid synthesis is accompanied by little apparent change in basal rates of lipolysis in vitro (61, 79) or in vivo (80), but a progressive increase in rates of intracellular reesterification of fatty acids (80). The degree to which responses to insulin of glucose-utilizing pathways, such as glycerogenesis, are truly enhanced in ruminant adipose tissue during mid and later lactation is not known. The question is of practical as well as academic interest because of the management implications of calorimetric observations that dairy cows may fatten more efficiently during midlate lactation than during the ensuing period when they are pregnant but not lactating (81). Some of the peripheral insulin resistance of early lactation can also be attributed to skeletal muscle, as

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shown by the attenuated response of hindlimb glucose uptake in vivo to low and high doses of insulin in lactating compared to nonlactating ewes (82). As in adipose tissue, the underlying mechanisms of insulin resistance appear to be postreceptor because lactation had no effect on number, affinity, or tyrosine kinase activity of insulin receptors in ovine (63) or caprine (83) skeletal muscle.

REGULATION AND COORDINATION OF METABOLIC ADAPTATIONS Concepts of Metabolic and Homeorhesis

Regulation—Homeostasis

The partitioning of glucose and other nutrients to various body tissues may be considered to involve two levels of regulation: homeostasis and homeorhesis. The term homeostasis was coined and formalized by Cannon (84) but the concept is identical with the idea of maintenance of a stable milieu interieur as postulated by Bernard (85). A classic example of homeostasis, first studied by Bernard, is the acute regulation of plasma glucose concentration by what we now know to be the reciprocal actions of the pancreatic hormones, insulin and glucagon, on glucose production and use in multiple tissues and organs. Insulin is an especially powerful mediator of many different regulatory effects, most of which serve to acutely maintain metabolic equilibrium in the face of short-term variations in nutrient supply and demand. This makes it a pivotal target in the mediation of chronic metabolic adaptations to pregnancy and lactation, including altered hepatic production and peripheral utilization of glucose. The concept of homeorhesis as it applies to regulation of nutrient partitioning was introduced by Bauman and Currie (86) and has been elaborated upon in numerous reviews by ourselves and others (e.g., 53, 87,88). Bauman and Currie defined homeorhesis as the "orchestrated or coordinated changes in metabolism of body tissues necessary to support a [dominant] physiological state". Notably, their examples were drawn almost exclusively from observations on pregnant and lactating ruminants. The postulated key features of homeorhesis are its chronic nature, i.e., hours or days vs. the seconds or minutes required for most examples of negative feedback regulation by homeostasis; its simultaneous influence on many tissues and organs, and functional systems (including the immune system); and its mediation through altered tissue responses to

homeostatic signals which may occur at various levels from ligand-receptor interactions through postreceptor signal transduction to downstream effects on transcription and posttranscriptional regulation of key enzymes and other regulatory factors. The latter two characteristics are especially evident in examples discussed in the next section.

Homeorhetic Regulation of Adaptations in Glucose Metabolism During Pregnancy and Lactation The extracellular nature of homeorhetic regulation implies its mediation by hormones and possibly, other humoral factors. Different hormones are likely to play predominant roles in mediating the altered responses to insulin and other homeostatic effectors during different phases of pregnancy and lactation. Numerous candidates have been suggested, some of which are listed in Table II. Unfortunately, with the notable exception of somatotropin and its pleiotropic influences on mammary and nonmammary tissues during lactation, consistent and conclusive evidence for the homeorhetic roles of most of these hormones has yet to be obtained. Metabolic responses to all of the hormones listed in Table II have been investigated in vivo and in vitro but inadequate experimental design may have produced some negative results. For example, simply measuring changes in concentrations or flux rates of blood metabolites within several hours of administering a putative homeorhetic agent might be insufficient to detect chronic, subtle influences on tissue metabolism. We have recently reviewed evidence that peripheral glucose utilization may be enhanced during midpregnancy and suppressed during late pregnancy by positive or negative effects of ovarian or placental hormones on insulin responses of target tissues (1). In general, the evidence for a positive effect of progesterone in mid pregnancy and negative effects of estradiol and placental lactogen in late pregnancy is hardly convincing, although the postulated roles of these hormones remain teleologically attractive. For example, chronic treatment of ovariectomized, nonpregnant ewes with estradiol-17(3 to mimic the high levels of circulating estradiol during late pregnancy causes a reduction in adipose lipogenesis and fatty acid esterification in vitro (89). However, when we treated ewes in a similar manner we were unable to establish a clear picture of altered peripheral responses to insulin using

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Table II. Possible Homeorhetic Hormones and Glucose-Related Tissue Responses in Pregnancy and Lactation State

Hormone

Putative action

Tissue/response

Mid pregnancy

Progesterone

f insulin sensitivity

T adipose glucose uptake T adipose lipogenesis

Late pregnancy

Placental lactogen Estrogens

4- insulin sensitivity and responsiveness

A glucose uptake by adipose and muscle 1 adipose lipogenesis T muscle glycolysis and lactate release

Lactogenesis, early lactation

Prolactin Estrogen Cortisol Somatotropin

1 insulin sensitivity and responsiveness

T liver gluconeogenesis 1 glucose uptake by adipose and muscle 1 adipose lipogenesis 1 protein synthesis -j T protein degradation I muscle T amino acid release J

the hyperinsulinemic, euglycemic clamp technique, despite persistent, superficial signs of modest insulin resistance (moderate hyperglycemia and hyperinsulinemia) in treated animals (90). Convincing evidence of a role for placental lactogen in the mediation of maternal insulin resistance during late pregnancy remains similarly elusive (1,91). Until the appropriate experiments are done, including chronic, in vivo administration of physiologically relevant amounts of pure native or recombinant hormone, support for this hypothesis will remain indirect and correlative. This includes our preliminary observation that the repartitioning of maternal glucose to favor the conceptus in moderately undernourished ewes (34) is associated with increased placental gene expression and maternal plasma concentrations of placental lactogen (R. A. Ehrhardt, R. V. Anthony, and A. W. Bell, unpublished). The various metabolic adaptations associated with lactogenesis and established lactation, described in preceding sections, are presumably influenced by the plethora of hormonal changes through this period. These include progesterone withdrawal and major increases in circulating levels of estradiol, cortisol and prolactin just before parturition, and elevated levels of somatotropin which are sustained from parturition through early lactation (49). Among these hormones, only somatotropin can be assigned a clear and powerful role in the homeorhetic regulation of adaptations in glucose production and disposal, coordinated with other metabolic changes in mammary and nonmammary tissues. Much of the evidence for these mechanisms has come from the large number of studies on effects of exogenous bovine somatotropin (bST) in lactating dairy cows and other ruminants. However, in

nonruminant species also, somatotropin may be a more important regulator of milk secretion than hitherto believed. For example, Flint and colleagues showed that in lactating rats treated with neutralizing antibody against somatotropin and(or) bromocriptine, somatotropin and prolactin probably act in concert, with certain of their individual effects revealed only in the absence of the other (see review, 69). Physiological responses to bST in lactating cows have been the subject of a recent, detailed review (92) so the rest of this section will focus mostly on subsequent, mechanistic studies of the actions of somatotropin on glucose metabolism in nonmammary tissues. These and previously documented effects of bST are summarized in Table III. Somatotropin treatment causes moderate increases in hepatic gluconeogenesis in lactating ruminants (see 92), concomitant with increased mammary glucose demands. This effect appears to involve a diminished ability of insulin to inhibit gluconeogenesis, as observed in glucose clamp studies of growing steers (93), and inferred from increased hepatic glucose production despite moderate hyperinsulinemia in lactating cows (94) treated with bST. However, this explanation is not entirely consistent with a previously discussed report of increased insulin suppression of glucose production in goats during early lactation (76), when plasma levels of endogenous somatotropin are presumably high. The importance of mammary glucose demand as a stimulus for hepatic gluconeogenesis is illustrated by the sluggish increase in glucose production of ewes artificially stimulated to lactate, associated with slowly increasing milk yield, compared to the brisk, pronounced postpartum increase in both responses in naturally lactating animals (95). It is per-

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274 haps pertinent that the combination of hormones used to induce lactation did not include somatotropin (95), unlike recent, more efficacious formulations used in dairy cows (R. S. Kensinger, personal communication). Somatotropin is a potent inhibitor of insulin-stimulated glucose utilization and lipogenesis in adipose tissue, as reviewed in detail elsewhere (92). It thus seems likely that elevated plasma levels of this hormone account for much of the pronounced insulin resistance of adipose tissue during early lactation. Somatotropin treatment, like early lactation, does not affect insulin binding or insulin-stimulated receptor kinase activity in ovine adipose tissue (96). The postreceptor locus of somatotropin's effects on insulin signaling may involve inhibition of PI3-kinase, as recently demonstrated in sheep adipose tissue (97). This component of one arm of the insulin signal transduction system appears to be involved in regulation of glucose transport (98) and lipogenesis (99) in rat adipocytes. It is therefore notable that chronic treatment of lactating dairy cows with bST caused a decrease from low to Table III. Effects of Bovine Somatotropin on Glucose Metabolism and Related Processes in Specific Tissues in Lactating Ruminants" Tissue Mammary

Liver

Adipose

Muscle

Pancreas

a b

Process T milk synthesis t lactose synthesis t glucose uptake 4> GLUT 1 mRNA t blood flow consistent with increase in milk yield T hepatic gluconeogenesis 1 ability of insulin to inhibit gluconeogenesis •I lipogenesis 4 ability of insulin to stimulate lipogenesis 1 glucose uptake 4- GLUT4 mRNA 4 glucose uptake T lactate output 1 glucose oxidation (inferred) i insulin receptor abundance and tyrosine kinase activityb 1 GLUT4 mRNA + basal or glucose-stimulated secretion of insulin + basal or insulin/glucose-stimulated secretion of glucagon

Adapted from (92). T = increased, i = decreased, + = no change. Demonstrated in nonlactating animals and consistent with observations on lactating animals.

undetectable levels of mRNA for GLUT4 in omental adipose tissue (65). Treatment with exogenous bST causes a reduction in glucose uptake by hindlimb muscle in lactating dairy cows (100) and growing steers (93). This is probably mediated by inhibition of the actions of insulin on glucose transport and metabolism because bST treatment caused reductions in insulin receptor protein abundance and tyrosine kinase activity in skeletal muscle of rapidly growing lambs (96), and in GLUT4 mRNA abundance in muscle of lactating cows (65). Although not examined directly, it seems likely that the bST-induced reduction in whole-body oxidation of glucose (101) is at least partly due to decreased oxidation in muscle. Somatotropin-induced insulin resistance may also influence mobilization of amino acids from skeletal muscle soon after parturition. For example, the antiproteolytic effect of insulin was significantly reduced in human subjects infused with somatotropin (102). This might seem paradoxical, considering the fact that bST treatment of growing steers increases muscle protein synthesis and deposition (103). However, the latter responses are almost certainly mediated by increased activity of the IGF system which, in periparturient (104) as in experimentally underfed, lactating cows (105), is unresponsive to somatotropin. In contrast to its inhibitory effect on GLUT4 mRNA expression in adipose tissue and muscle, bST treatment had no effect on abundance of GLUT1 mRNA or protein in mammary tissue of lactating cows (65). These authors argued that since bST has little effect on plasma glucose concentration in lactating cows (101), increased mammary uptake of glucose must be determined by increased mammary blood flow. The fact that mammary blood flow is increased by bST (or, for that matter, any treatment that enhances milk yield) is undeniable (see 92). However, we believe that increased mammary perfusion is simply a coordinated response to increased mammary metabolic activity, including increased glucose utilization for lactose synthesis, rather than a primary regulatory factor. The observation that increasing systemic glucose supply by postruminal infusion had no effect on milk or lactose yield of control or bST-treated cows fed a wellbalanced basal ration (106) is consistent with this notion. It is evident that treatment of lactating ruminants with exogenous somatotropin has manifold, coordinated effects on glucose metabolism in liver, adipose tissue, skeletal muscle, and mammary gland. These,


together with the hormone's ability to effect some of these responses by altering tissue responses to insulin, are entirely consistent with its postulated homeorhetic role (92). When these features are considered together with its pattern of endogenous secretion in untreated ruminants, a powerful case can be made for the importance of somatotropin as the principal homeorhetic regulator of glucose partitioning between the mammary gland and key nonmammary tissues involved in glucose production and utilization.

CONCLUSIONS Late pregnancy and lactation share common characteristics in the regulation of glucose metabolism in that each requires significant metabolic adaptations in various nonuterine or nonmammary tissues to meet the predominant glucose requirements of the gravid uterus or lactating mammary gland. These multiple adaptations evolve in chronic fashion, and in each state are at least partly mediated by development of insulin resistance in maternal peripheral tissues. Such characteristics are the hallmarks of homeorhetic regulation of nutrient partitioning (86), implying the modulating influence of chronically-acting, extracellular regulatory factors. The identity of the factor(s) responsible for homeorhetic control of glucose metabolism during pregnancy remains to be established. In contrast, there is persuasive evidence that somatotropin has a primary role in this and many other facets of the regulation of nutrient disposal and metabolism during lactation.

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