NEW OPPORTUNITIES FOR MANAGING METABOLISM IN TRANSITION COWS. K. L. Smith and T. R. Overton Department of Animal Science Cornell University

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1 NEW OPPORTUNITIES FOR MANAGING METABOLISM IN TRANSITION COWS K. L. Smith and T. R. Overton Department of Animal Science Cornell University Research on the biology and management of transition dairy cows has received substantial emphasis during the past 15 years (reviewed by Overton and Waldron, 24). Despite this large quantity of research and concurrent focus on application of management strategies within the dairy industry to improve transition cow health and performance, many dairy farms continue to struggle with high rates of metabolic disorders in fresh cows. Some of the ongoing issues relate to problems with application of existing knowledge on a consistent basis on individual farms. However, it is also possible that there are fundamental aspects of metabolism and potential application of management strategies to alter metabolism of transition cows to lower their risk of developing metabolic disorders that remain undiscovered and unrealized. Our group has focused a significant part of our research effort during the past several years on such a possibility. It is well-recognized that the dairy cows undergo important metabolic adaptations during late pregnancy to support fetal demands and at the onset of lactation to support milk production. These homeorhetic adaptations involved in the regulation of nutrient and energy partitioning during late pregnancy and early lactation occur in a variety of target tissues, and typically involve changes in responses of tissues such as adipose tissue and muscle to homeostatic signals such as insulin and epinephrine (Bauman and Currie, 198; Bell, 1995). One major adaptation includes a large increase in glucose demand by the mammary gland that is supported by a decrease in oxidation of glucose by peripheral tissues (Bennink et al., 1972) and an increase in glucose output by the liver (Reynolds et al., 23). Additionally, the mobilization of body fat to meet overall energy demands is supported by a decrease in the response of adipose tissue to insulin (Petterson et al., 1993, 1994). The net result of these adaptations is coordinated support of fetal needs and subsequent high milk production in the face of decreasing and eventually insufficient dry matter intake (DMI) during late pregnancy and early lactation. The success of this metabolic coordination influences productive performance during the ensuing lactation, the risk of developing metabolic disorders during early lactation, and subsequently reproductive capacity. These coordinated adaptations are regulated by changes in expression of many genes during the transition period that eventually result in changes in endocrine signaling in various tissues of the cow. Although the homeorhetic adaptations involved during the transition period are generally understood (Bauman and Currie, 198; Bell, 1995), the knowledge of the timing of these changes as the cow goes through the transition period is not well understood. By further characterizing the changes in tissue responses to homeostatic signals, it may reveal the potential to develop management strategies to modulate metabolism during the transition period.

2 One of the primary homeostatic signals regulating metabolism is insulin. During the prepartum period, homeorhetic controls facilitate insulin resistance, which contributes to glucose sparing to meet the glucose demands of the gravid uterus. Insulin resistance exists whenever a greater than normal concentration of insulin is required to elicit a normal response (Kahn, 1978). Peripartal insulin resistance manifests itself as both decreased sensitivity (Petterson et al., 1993) and decreased responsiveness of tissues to insulin (Petterson et al., 1994). In addition, insulin is less able to promote lipogenesis and suppress lipolysis in adipose tissue during the prepartum period (Bell, 1995) and this phenomenon continues into early lactation. Leury et al. (23), using the hyperinsulinemic euglycemic clamp technique in dairy cattle, reported that insulin resistance begins prior to parturition and continues into early lactation. Recent research conducted in our laboratory utilizing the glucose tolerance test (Smith, 24) suggested that insulin sensitivity with regard to glucose metabolism may actually be greater on day 5 and 28 postpartum compared with day 1 before expected parturition, but it is likely that this interpretation is confounded by the large increase in non-insulin dependent glucose utilization presented by the mammary gland upon lactation (Figure 1). In addition, despite the large increase in circulating insulin concentrations, the slope of NEFA decrease following glucose administration was significantly less on day -1 compared to the postpartum periods of study (Figure 2). These results provide a potential biological explanation for the interesting findings of Burhans et al. (1997), who determined that propylene glycol administration was effective in decreasing circulating NEFA concentrations during the postpartum period; however, circulating NEFA concentrations were refractory to propylene glycol administration during the prepartum period. Glucose area, mg/dl a b b Day -1 Day +5 Day +28 Insulin area, ng/ml a b b Day -1 Day +5 Day Figure 1. Glucose (left side) and insulin responses (right side) of cows administered glucose tolerance tests on 1 d prior to expected parturition and 5 and 28 d postpartum (Smith, 24).

3 25 b Day -1 Day +5 NEFA slope, ueq/l x min a c Day +28 Figure 2. Slope of the NEFA decrease of cows administered glucose tolerance tests on 1 d prior to expected parturition and 5 and 28 d postpartum (Smith, 24). In a subsequent experiment, we evaluated changes in tissue responsiveness and sensitivity to insulin throughout the lactation cycle by using responses to multiple doses of insulin in an insulin challenge model. As expected following insulin challenge (Table 1), plasma glucose decreased and plasma insulin increased dose-dependently in all groups of cows. After challenge with.8 μg insulin/kg of body weight (BW), glucose area under the curve (AUC) was lowest in both prepartum groups and insulin AUC was highest in the groups challenged during d -12 to -8 and during the early postpartum period, suggesting that insulin sensitivity was lowest during the immediate prepartum period. In response to 1.6 μg insulin/kg of BW, glucose AUC was highest in cows challenged during the midlactation (ML) period while cows challenged during the periparturient period responded comparably. In addition, insulin area under the curve was highest for cows challenged during d -12 to -8 and d +3 to +7. These data suggest that midlactation cows have the greatest responses to insulin and that cows challenged during d -12 to d -8 may be the most resistant to insulin. Table 1. Responses to insulin challenges (Smith et al., 26b). Day relative to parturition -3 to to to to +3 ML SE Glucose AUC.8 μg/kg BW 148 c 194 bc 238 a 211 ab 24 a μg/kg BW 249 b 254 b 256 b 246 b 358 a 35 Insulin AUC.8 μg/kg BW 74 b 155 a 136 a 124 a 53 b μg/kg BW 19 b 269 a 223 ab 196 b 172 b 28 Means within the same row with different superscripts differ, P <.5

4 As mentioned above, insulin resistance is a necessary and important adaptation that develops during late pregnancy and continues into early lactation in order to spare glucose for the fetus and mammary gland. However, we believe that insulin resistance also is a major contributor to the concurrent increase in circulating NEFA and decrease in DMI that characteristically occurs during the last 7 to 1 days before calving (Figure 3). This relationship is associated with all periparturient health disorders with etiologies based in energy metabolism, immune function, or both (displaced abomasum, ketosis, fatty liver, retained placenta, metritis, and mastitis). DMI (kg/d) DMI NEFA NEFA, ueq/l Day relative to calving Figure 3. Relationship between DMI and plasma NEFA concentration during the periparturient period (Smith, 24). An increasing body of evidence suggests that plane of nutrition, in particular energy intake during the prepartum period, modulates the degree of insulin resistance and hence the relationships between NEFA and DMI during the immediate peripartal period. Mashek and Grummer (23) reported that cows that had larger decreases in DMI during the prepartum period, generally because of higher DMI during weeks 3 and 4 before calving, had higher concentrations of plasma NEFA and liver triglycerides during the postpartum period. More direct experimental evidence was provided by Douglas et al. (26), who reported that cows fed at 8% of calculated energy requirements for the entire dry period had lower NEFA concentrations during the postpartum period, lower concentrations of both circulating glucose and insulin during the prepartum period, and higher DMI during the postpartum period than cows consuming 16% of predicted energy requirements throughout the dry period. In addition, Holtenius et al. (23) determined that cows that were dramatically overfed (178% of calculated energy requirements) for the last 8 weeks before calving had higher concentrations of insulin and glucose during the prepartum period, greater insulin responses to glucose challenge during the prepartum period, and higher concentrations of circulating NEFA during the postpartum period than cows fed for 75 or 11% of calculated energy requirements. Furthermore, Agenas et al. (23) reported that the same cows fed for 178% of calculated energy requirements prepartum had lower DMI and prolonged negative energy balance during the postpartum period compared with cows assigned to

5 the other two prepartum treatments. Recently, Dann et al. (26) demonstrated that overfeeding (15% of calculated energy requirements) during the far-off period may have exacerbated insulin resistance as cows approached calving, resulting in higher NEFA and BHBA and lower DMI and energy balance during the first 1 days postcalving. This knowledge has led to an evolution in recommendations for energy nutrition of dairy cows during both the far-off and close-up periods during the past several years, with the goal of meeting, but not dramatically exceeding energy requirements. In practice, this can be achieved by formulating diets during the far-off period to contain no more than.59 to.63 Mcal/lb of NE L. During the close-up period, conventional recommendations have been to maximize DMI, and hence energy intake. Although this still applies in many herd situations, we believe that some well-managed herds in which close-up cows consume large amounts of feed (> 3 to 32 lb/day of dry matter) have increased rates of metabolic disorders because of excessive energy intake during the close-up period. Accordingly, some of these herds have had success in moderating energy intake during the close-up period in group-feeding situations by incorporating straw or other low potassium, low energy forage to lower overall dietary energy concentration (.66 to.68 Mcal/lb of NE L ) and ensure adequate, but not excessive energy intake within the dynamics of group-feeding and competition among animals. Although the specific biological regulator for accentuating effects of overfeeding on insulin resistance has not been elucidated, we speculate that most aspects of insulin resistance in adipose tissue likely are mediated through the action of peroxisome proliferator activated receptor-gamma (PPARγ) in dairy cows during the transition period. Peroxisome proliferator activated receptors (PPAR) are a family of nuclear receptors that regulate gene expression in many tissues in response to ligand binding and are responsible for a wide array of regulatory functions in mammals. One member of the PPAR family, PPARα, is known to regulate aspects of hepatic fatty acid oxidation (Berger et al., 25). As mentioned above, PPARγ, has been characterized in bovine tissue and is highly expressed in bovine adipose tissue (Sundvold et al., 1997). Activation of PPARγ has been shown to potentiate adipocyte differentiation and stimulate insulin action, while decreasing the release of free fatty acids from the adipocyte (Guo and Tabrizchi, 26; Houseknecht et al., 22). We believe that PPARγ is a logical target for management strategies to selectively modulate insulin resistance in the periparturient dairy cow and fundamentally decrease risk of metabolic disorders during the transition period. Although we speculate that dramatic overfeeding during the prepartum period may exacerbate insulin resistance and accentuate the changes in DMI and plasma NEFA concentrations during the peripartal period as described above through actions on PPARγ, even cows that are not overfed are at risk for developing health disorders because of decreased DMI and increased plasma NEFA. Therefore, the prospect of fundamentally altering the dynamics of DMI and plasma NEFA in transition cows by modulating PPARγ using direct ligands is very attractive.

6 There are many natural ligands for PPARγ including fatty acids and prostaglandins (Houseknecht et al., 22), but the most potent ligands are synthetic and include the thiazolidinediones (TZD). Administration of TZD to animal models of non-insulin dependent diabetes mellitus (NIDDM) increased insulin action, decreased plasma free fatty acids and improved pancreatic β-cell function. Administration of TZD to late pregnant rats significantly lowered plasma triglyceride concentrations (Sevillano et al., 25) and Larsen et al. (23) reported a significant increase in food intake in rats given TZD over an extended period of time. Limited research has been conducted on the activation of PPARγ and the administration of synthetic PPARγ ligands, specifically TZD, to ruminant species. Kushibiki et al. (21) determined that administration of TZD to steers injected with tumor necrosis factor alpha (TNFα) in order to induce an insulin resistant state had decreased plasma concentrations of NEFA, insulin, and glucagon suggesting administration of TZD restores insulin action by reducing circulating NEFA. The authors concluded that TZD administration reversed insulin resistance caused by experimental activation of the immune system. We hypothesized that activation of PPARγ through administration of TZD to dairy cows during the prepartum period would modulate specific aspects of insulin resistance in adipose tissue and decrease circulating concentrations of NEFA during the periparturient period, which would potentially alter the dynamics of DMI during the peripartal period. Therefore, the objectives of our experiment were to determine the metabolic effects during the periparturient period of TZD administration during the late prepartum period. The Cornell University Institutional Animal Care and Use Committee approved all procedures involving animals. Holstein cows (n = 14) entering their second or later lactation were utilized in this experiment. Five cows were removed from the study prior to analysis for the following reasons: one cow was not pregnant, one cow pinched a nerve at calving, two cows retained their placentas (RP) and one cow had a uterine torsion. All cows were dried off 4 to 6 d prior to their expected parturition dates. Treatments were balanced for BCS and previous ME35 milk yield. Cows were moved to individual tiestalls at approximately 32 days before expected parturition and housed there through 8 d postpartum. Cows were administered either 2,4 TZD (4. mg/kg BW) or saline (control) once daily by intrajugular infusion once daily from 25 days before expected parturition until parturition. Cows were fed a TMR during the prepartum period to provide no more than 13% of predicted energy requirements. During the postpartum period cows were fed a common TMR for ad libitum intake. Fresh feed was provided each morning at 1 h, Orts were weighed and recorded daily and water was made available at all times. Body weights and body condition scores of each animal were recorded on one day each week throughout the study. Daily observations, daily temperatures and general health records were maintained throughout the study. After parturition cows were

7 milked were twice daily according to established procedures at the Cornell University Teaching and Research Center for the first 8 days in milk and then switched to three times a day milking. Yields were recorded daily for the first 3 days in milk, milk was sampled once on day 8 postpartum and composition was determined. Results were used in the calculation of energy balance (NRC, 21). Cows were fitted with an indwelling jugular catheter on day 27 before expected parturition, and blood samples were collected once daily at 13 h via the catheter from d 26 before expected parturition to d 8 postpartum. Insulin challenges (.8 and 2.4 μg/kg BW) were administered at 14 h on day 1 and 9 relative to expected parturition, and on day +6 and +7 postpartum. Blood was sampled at 3, 2, 1,, 2.5, 5, 7.5, 1, 12.5, 15, 17.5, 2, 25, 3, 45, 6, 9, 12, 125, and 13 min relative to the insulin injection. Plasma was harvested and concentrations of glucose, NEFA, insulin, and beta-hydroxybutyrate (BHBA) were determined. Liver samples were taken on day 8 postpartum and analyzed for triglyceride and glycogen content. All cows were then returned to the herd. Following insulin challenges, response patterns of insulin and glucose were similar at all doses of insulin. The nadir for plasma concentrations of glucose occurred by 45 min following insulin administration and returned to baseline by 12 min. To minimize the contribution of clearance and counterregulatory effects, the response area for glucose was calculated from the time of challenge to 25 min postchallenge and the response area for insulin was calculated from the time of challenge to 3 min postchallenge. Response areas were corrected for differences in baseline concentrations (mean of concentrations at -3, -2, -1, and min from the time of the challenge injection for glucose and mean of concentrations at -3, -2, and -1 min from the time of the challenge injection for insulin). Plasma glucose and insulin responses to.8 and 2.4 μg insulin/kg of BW were used as proxies for tissue sensitivity and responsiveness, respectively. Metabolite and hormone responses to each challenge were calculated as area under the response curve using the EXPAND procedure in SAS (SAS, 21) and then subjected to analysis of variance using the MIXED procedure of SAS (21). The model included the effects of treatment. Pretreatment values for NEFA, glucose, insulin, BHBA, DMI, BW, and BCS were used as covariates during analysis of covariance applied to their corresponding measurements during the treatment period and data from the pre- and postpartum periods were analyzed together. Milk yield values were reduced to weekly means to calculate yields of milk components (NRC, 21). Prepartum and postpartum energy balance was calculated based on equations provided in the NRC (21). Analysis of variance was conducted on plasma concentrations of NEFA, glucose, insulin, BHBA, DMI and milk yield using the MIXED procedure of SAS (21) for a completely randomized design with repeated measures. The model included effects of covariate, treatment, time, and the interaction of treatment and time. Least squares means are reported throughout, and significance was declared at P <.5 and trends were declared at.5 < P <.1.

8 Plasma NEFA concentrations (Figure 4) were similar for cows assigned to the two treatments during much of the prepartum period, but were decreased at parturition and during the first 7 days postpartum for cows administered TZD during the prepartum period (treatment by time, P <.1). Cows administered TZD tended (P =.9) to have lower plasma NEFA throughout the entire periparturient period compared to those administered saline prepartum. Plasma glucose, insulin and BHBA concentrations were not affected (P >.1) by treatment. There was no effect (P >.1) of prepartum treatment on liver glycogen or triglyceride concentrations (Figure 5). Plasma NEFA (ueq/l) TZD CTL Day relative to parturition Figure 4. Pattern of plasma NEFA concentrations during the periparturient period for cows administered TZD or saline during the prepartum period. Values are least squares means, n = 4 for TZD and n = 5 for saline; treatment by time, P <.1 (Smith et al., 26a) Liver triglycerides, % wet weight TZD CTL Liver glycogen, % wet weight TZD CTL Figure 5. Concentrations of liver triglycerides (left side) and glycogen (right side) on day 8 postpartum for cows administered TZD or saline prepartum. Values are least squares means, n = 4 for TZD and n = 5 for saline; P >.1 (Smith et al., 26a).

9 Average DMI was greater for cows administered TZD compared to controls (14.8 vs kg/d; P <.5). There was no treatment by time interaction (P >.1; Figure 6) for DMI as both groups of cows consumed the same DMI prepartum; however, the TZDtreated cows consumed substantially more DM postpartum, resulting in the overall effect of treatment on DMI. Cows assigned to the TZD and control treatments consumed 128 and 125% of predicted energy requirements, respectively, during the prepartum period. Cows administered TZD had lower milk yield than controls during the immediate (day and day 1) postpartum period but then produced more milk than controls through 8 days postpartum (Figure 7; treatment by time, P <.1). Differences in milk component concentrations measured on day 8 postpartum were not significant (P >.1). There was no effect (P >.1) of prepartum TZD treatment on milk yield measured from day through day 3 postpartum. Saline treated cows tended (P =.1) to have higher BCS (3.2 vs. 3.) than TZD-treated cows, but there was no effect (P >.1) of prepartum treatment on BW. Finally, there was no effect (P >.1) of prepartum treatment on calf birth weight (average 45 kg) or calculated net energy balance on day 7 postpartum (-6.4 vs -3.9 Mcal for control and TZD-treated cows, respectively) TZD CTL DMI (kg/d) Day relative to parturition Figure 6. Pattern of DMI during the periparturient period for cows administered TZD or saline. Values are least squares means, n = 4 for TZD and n = 5 for saline; effect of treatment, P <.5 (Smith et al., 26a).

10 6 5 TZD CTL Milk yield (kg/d) X 3X Day postpartum Figure 7. Pattern of milk yield during the first 3 days postpartum for cows administered TZD or saline. Values are least squares means, n = 4 for TZD and n = 5 for saline; P-value for the interaction of treatment and day during the 2X period was.1 and.31 from day through day 3 postpartum (Smith et al., 26a). Responses to pre- and postpartum insulin challenges were similar within physiological state. With insulin administration, the plasma concentration of glucose started to decrease immediately at time zero, reached minimum by 45 min post injection in all groups at all doses, and returned to baseline by 12 min post injection. Prepartum treatment with TZD did not affect (P >.1) the glucose or insulin AUC for both doses of insulin,.8 μg/kg BW and 2.4 μg/kg BW, during both the pre- and postpartum periods. Thiazolidinediones administered to cows in our experiment did not appear to have an effect on insulin-dependent glucose utilization by muscle, hence TZD administration did not appear to compromise the important adaptations in glucose metabolism described at the beginning of this paper. This result is expected, as PPARγ expression is low in skeletal muscle (Sundvold et al., 1997), which represents the predominant target for insulin-dependent glucose utilization. SUMMARY, CONCLUSIONS, AND IMPLICATIONS Insulin resistance in late pregnancy is an important adaptation for the onset of lactation in dairy cows, particularly with regard to glucose metabolism. However, insulin resistance prepartum likely also contributes to the mobilization of NEFA beginning before calving that predisposes cows to dramatic declines in DMI observed before calving. Selective modulation of insulin resistance during the prepartum period may decrease the NEFA spike and improve periparturient performance and health. Although moderating energy intake before calving in situations of dramatic overfeeding may prevent diet-accentuated insulin resistance, pharmaceutical intervention appears to be a more potent and robust means of selectively modulating insulin resistance. In the first study using pharmaceutical intervention to reduce prepartum insulin resistance that was reported in part in this text, cows administered TZD during the

11 prepartum period had dramatically lower plasma NEFA concentrations than controls immediately prepartum and through 7 days postpartum (Smith et al., 26a). This was likely due to the direct effect of PPARγ activation and possibly due to changes in genes involved in lipid storage. Prepartum TZD administration increased postpartum DMI, possibly due to the lower plasma NEFA concentrations in these cows. Although there were small, but statistically significant interactions for milk yield during the first few days of lactation, cows assigned to both treatments were producing similar amounts by 2 weeks postcalving. We are conducting larger-scale experimentation to more fully evaluate effects of prepartum TZD administration on performance. In conclusion, administration of TZD during the late prepartum period has the potential to substantially improve metabolic health and production of transition dairy cows by moderating the dramatic increase in NEFA and decrease in DMI during the immediate peripartal period. These findings have substantial potential implications for reducing the risk for energy- and immune-related metabolic disorders in transition dairy cows. There are numerous opportunities for future research in this area including the investigation of dose-response effects, delivery methods such as rumen-protected supplements and controlled-release devices, the potential of using more potent TZDs or similar compounds, the use of dual agonists that target both PPARγ and PPARα, or administration for a shorter amount of time during the prepartum period. ACKNOWLEDGEMENTS The authors gratefully acknowledge Amy Rauf, Sarah Stebulis, Suzanne Stachnik, Erin Morgan, Betsy Johnson, Amy Kulick, Greg Johnson, Matt Waldron, James Perfield II, Ramona Ehrhardt, Betsy Thering, Jose Manuel Ramos-Nieves, Debra Dwyer, and the staff at the Cornell Teaching and Research Dairy Center. REFERENCES Agenas, S., E. Burstedt, and K. Holtenius. 23. Effects of feeding intensity during the dry period. 1. Feed intake, body weight, and milk production. J. Dairy Sci. 86: Bauman, D. E., and W. B. Currie Partitioning of nutrients during pregnancy and lactation: a review of mechanisms involving homeostasis and homeorhesis. J. Dairy Sci. 63: Bell, A. W Regulation of organic nutrient metabolism during transition from late pregnancy to early lactation. J. Anim Sci. 73: Bennink, M. R., R. W. Mellenberger, R. A. Frobish and D. E. Bauman Glucose oxidation and entry rate as affected by the initiation of lactation. J. Dairy Sci. 55:712 (Abstr.). Berger, J. P., T. E. Akiyama, and P. T. Meinke. 25. PPARs: therapeutic targets for metabolic disease. Trends Pharmacol. Sci. 26: Burhans, W. S., E. A. Briggs, J. A. Rathmacher, and A. W. Bell Glucogenic supplementation does not reduce body tissue protein degradation in periparturient dairy cows. J. Dairy Sci. 8(Suppl. 1):167. (Abstr.)

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