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Effect of hormonal and energy-related factors on plasma adiponectin in transition dairy cows

Open ArchivePublished:August 23, 2017DOI:https://doi.org/10.3168/jds.2017-13274

      ABSTRACT

      In transition dairy cows, plasma levels of the insulin-sensitizing hormone adiponectin fall to a nadir at parturition and recover in early lactation. The transition period is also characterized by rapid changes in metabolic and hormonal factors implicated in other species as positive regulators of adiponectin production (i.e., negative energy balance, lipid mobilization) and others as negative regulators (i.e., reduced leptin and insulin and increased growth hormone and plasma fatty acids). To assess the role of onset of negative energy balance and lipid mobilization after parturition, dairy cows were either milked thrice daily (lactating) or never milked (nonlactating) for up to 4 wk after parturition. Plasma adiponectin was 21% higher across time in nonlactating than lactating cows. Moreover, nonlactating cows recovered plasma adiponectin at similar rates as lactating cows even though they failed to lose body condition. Next, we assessed the ability of individual hormones to alter plasma adiponectin in transition dairy cows. In the first experiment, dairy cows received a constant 96-h intravenous infusion of either saline or recombinant human leptin starting on d 8 of lactation. In the second experiment, dairy cows were studied in late pregnancy (LP, starting on prepartum d −31) and again in early lactation (EL, starting on d 7 postpartum) during a 66-h period of basal sampling followed by 48 h of hyperinsulinemic-euglycemia. In the third experiment, cows were studied either in LP (starting on d −40 prepartum) or EL (starting on d 7 postpartum) during a 3-h period of basal sampling followed by 5 d of bovine somatotropin treatment. Plasma adiponectin was reduced by an average of 21% in EL relative to LP in these experiments, but neither leptin, insulin, or growth hormone treatment affected adiponectin in LP or EL. Finally, the possibility that plasma fatty acids repress plasma adiponectin was evaluated by intravenous infusion of a lipid emulsion in nonpregnant, nonlactating cows in the absence or presence of glucagon for 16 consecutive hours. The intralipid infusion increased plasma fatty acid concentration from 102 to over 570 µM within 3 h but had no effect on plasma adiponectin irrespective of presence or absence of glucagon. Overall, these data suggest that energy balance around parturition may regulate plasma adiponectin but do not support roles for lipid mobilization or sustained changes in the plasma concentration of leptin, insulin, growth hormone, or fatty acids.

      Key words

      INTRODUCTION

      Adiponectin is a 30-kDa protein hormone synthesized exclusively by adipose tissue (
      • Kadowaki T.
      • Yamauchi T.
      • Kubota N.
      • Hara K.
      • Ueki K.
      • Tobe K.
      Adiponectin and adiponectin receptors in insulin resistance, diabetes, and the metabolic syndrome.
      ;
      • Wang Z.V.
      • Scherer P.E.
      Adiponectin, the past two decades.
      ). Adiponectin circulates as a homomultimer consisting of low molecular weight, medium molecular weight, and high molecular weight complexes containing 3, 6, or 18 or more adiponectin monomers (
      • Wang Y.
      • Lam K.S.L.
      • Yau M.
      • Xu A.
      Post-translational modifications of adiponectin: mechanisms and functional implications.
      ;
      • Wang Z.V.
      • Scherer P.E.
      Adiponectin, the past two decades.
      ). Adiponectin signals through 2 membrane-bound receptors, adiponectin receptor 1 and adiponectin receptor 2, which are found in most tissues including liver, muscle, and adipose tissue (
      • Kadowaki T.
      • Yamauchi T.
      • Kubota N.
      • Hara K.
      • Ueki K.
      • Tobe K.
      Adiponectin and adiponectin receptors in insulin resistance, diabetes, and the metabolic syndrome.
      ). Many lines of evidence suggest that adiponectin is an insulin sensitizer. First, decreased plasma adiponectin is observed in conditions and diseases characterized by insulin resistance (IR) such as obesity and type 2 diabetes (
      • Kadowaki T.
      • Yamauchi T.
      • Kubota N.
      • Hara K.
      • Ueki K.
      • Tobe K.
      Adiponectin and adiponectin receptors in insulin resistance, diabetes, and the metabolic syndrome.
      ;
      • Wang Z.V.
      • Scherer P.E.
      Adiponectin, the past two decades.
      ). This inverse relation between IR and plasma adiponectin has been found not only in humans but also in various animal models ranging from nonhuman primates to dolphins (
      • Hotta K.
      • Funahashi T.
      • Bodkin N.L.
      • Ortmeyer H.K.
      • Arita Y.
      • Hansen B.C.
      • Matsuzawa Y.
      Circulating concentrations of the adipocyte protein adiponectin are decreased in parallel with reduced insulin sensitivity during the progression to type 2 diabetes in rhesus monkeys.
      ;
      • Venn-Watson S.
      • Smith C.R.
      • Stevenson S.
      • Parry C.
      • Daniels R.
      • Jensen E.
      • Cendejas V.
      • Balmer B.
      • Janech M.
      • Neely B.A.
      • Wells R.
      Blood-based indicators of insulin resistance and metabolic syndrome in bottlenose dolphins (Tursiops truncatus).
      ). Second, administration of recombinant adiponectin improved insulin action in various mouse models of IR (diet-induced obese, lipoatrophic, db/db, and KKAy;
      • Kadowaki T.
      • Yamauchi T.
      • Kubota N.
      • Hara K.
      • Ueki K.
      • Tobe K.
      Adiponectin and adiponectin receptors in insulin resistance, diabetes, and the metabolic syndrome.
      ). Third, mutations leading to adiponectin loss of function promote IR in both mice and humans (
      • Kadowaki T.
      • Yamauchi T.
      • Kubota N.
      • Hara K.
      • Ueki K.
      • Tobe K.
      Adiponectin and adiponectin receptors in insulin resistance, diabetes, and the metabolic syndrome.
      ), whereas adiponectin overexpression in ob/ob mice is sufficient to normalize their excessive circulating levels of glucose and insulin (
      • Kim J.-Y.
      • van de Wall E.
      • Laplante M.
      • Azzara A.
      • Trujillo M.E.
      • Hofmann S.M.
      • Schraw T.
      • Durand J.L.
      • Li H.
      • Li G.
      • Jelicks L.A.
      • Mehler M.F.
      • Hui D.Y.
      • Deshaies Y.
      • Shulman G.I.
      • Schwartz G.J.
      • Scherer P.E.
      Obesity-associated improvements in metabolic profile through expansion of adipose tissue.
      ).
      We have demonstrated that the plasma concentration of adiponectin varies in a quadratic manner in transition dairy cows with the highest levels in late pregnancy (LP), a nadir on the day of parturition, and a progressive return to LP values over the first few weeks of lactation (
      • Giesy S.L.
      • Yoon B.
      • Currie W.B.
      • Kim J.W.
      • Boisclair Y.R.
      Adiponectin deficit during the precarious glucose economy of early lactation in dairy cows.
      ;
      • Mielenz M.
      • Mielenz B.
      • Singh S.P.
      • Kopp C.
      • Heinz J.
      • Häussler S.
      • Sauerwein H.
      Development, validation, and pilot application of a semiquantitative Western blot analysis and an ELISA for bovine adiponectin.
      ). This adiponectin profile occurs in parallel with the development of IR and the onset of negative energy balance (NEB) and coincides with rapid reduction in plasma insulin and leptin and reciprocal changes in plasma growth hormone (GH) and fatty acids (
      • Block S.S.
      • Butler W.R.
      • Ehrhardt R.A.
      • Bell A.W.
      • Van Amburgh M.E.
      • Boisclair Y.R.
      Decreased concentration of plasma leptin in periparturient dairy cows is caused by negative energy balance.
      ;
      • Rhoads R.P.
      • Kim J.W.
      • Leury B.J.
      • Baumgard L.H.
      • Segoale N.
      • Frank S.J.
      • Bauman D.E.
      • Boisclair Y.R.
      Insulin increases the abundance of the growth hormone receptor in liver and adipose tissue of periparturient dairy cows.
      ;
      • Giesy S.L.
      • Yoon B.
      • Currie W.B.
      • Kim J.W.
      • Boisclair Y.R.
      Adiponectin deficit during the precarious glucose economy of early lactation in dairy cows.
      ). Evidence in rodents and humans indicates involvement of these factors in regulating adiponectin production (
      • Delporte M.-L.
      • El Mkadem S.A.
      • Quisquater M.
      • Brichard S.M.
      Leptin treatment markedly increased plasma adiponectin but barely decreased plasma resistin of ob/ob mice.
      ;
      • Bobbert T.
      • Weicht J.
      • Mai K.
      • Möhlig M.
      • Pfeiffer A.F.H.
      • Spranger J.
      Acute hyperinsulinaemia and hyperlipidaemia modify circulating adiponectin and its oligomers.
      ;
      • Lubbers E.R.
      • List E.O.
      • Jara A.
      • Sackman-Sala L.
      • Cordoba-Chacon J.
      • Gahete M.D.
      • Kineman R.D.
      • Boparai R.
      • Bartke A.
      • Kopchick J.J.
      • Berryman D.E.
      Adiponectin in mice with altered GH action: links to insulin sensitivity and longevity?.
      ), but whether they contribute to variation in plasma adiponectin in transition dairy cows remains unknown.
      The objectives of the present study were to determine first whether the onset of NEB around parturition contributes to reduced plasma adiponectin and second whether sudden changes in the plasma concentrations of leptin, insulin, GH, or fatty acids regulate plasma adiponectin. Our results demonstrate that plasma adiponectin is sensitive to changes in energy balance in the immediate post-periparturient period but rule out leptin, insulin, GH, or fatty acids as factors contributing to reduced plasma adiponectin in transition dairy cows.

      MATERIALS AND METHODS

      Animals and Design

      Samples analyzed were from 5 previous experiments designed to identify effects of variation in energy balance, leptin, insulin, GH, or fatty acids (
      • Block S.S.
      • Butler W.R.
      • Ehrhardt R.A.
      • Bell A.W.
      • Van Amburgh M.E.
      • Boisclair Y.R.
      Decreased concentration of plasma leptin in periparturient dairy cows is caused by negative energy balance.
      ;
      • Leury B.J.
      • Baumgard L.H.
      • Block S.S.
      • Segoale N.
      • Ehrhardt R.A.
      • Rhoads R.P.
      • Bauman D.E.
      • Bell A.W.
      • Boisclair Y.R.
      Effect of insulin and growth hormone on plasma leptin in periparturient dairy cows.
      ;
      • Schoenberg K.M.
      • Giesy S.L.
      • Harvatine K.J.
      • Waldron M.R.
      • Cheng C.
      • Kharitonenkov A.
      • Boisclair Y.R.
      Plasma FGF21 is elevated by the intense lipid mobilization of lactation.
      ;
      • Ehrhardt R.A.
      • Foskolos A.
      • Giesy S.L.
      • Wesolowski S.R.
      • Krumm C.S.
      • Butler W.
      • Quirk S.
      • Waldron M.R.
      • Boisclair Y.R.
      Increased plasma leptin attenuates adaptive metabolism in early lactating dairy cows.
      ;
      • Caixeta L.S.
      • Giesy S.L.
      • Krumm C.S.
      • Perfield 2nd, J.W.
      • Butterfield A.
      • Schoenberg K.M.
      • Beitz D.C.
      • Boisclair Y.R.
      Effect of circulating glucagon and free fatty acids on hepatic FGF21 production in dairy cows.
      ). All experiments were performed in multiparous Holstein cows at Cornell University and were approved by the Cornell Institutional Animal Care and Use Committee. Procedures common to all experiments included housing in individual stalls and blood collection from chronic intrajugular catheters. Blood was processed to plasma by the addition of sodium heparin (15 IU/mL) and centrifugation. Unless otherwise mentioned, cows were fed unlimited amounts of TMR using automatic feeders and milked daily at 0600 and 1800 h after parturition. Experiments and associated specific procedures were as follows.

      Effect of the Periparturient Period and Leptin

      The study of
      • Ehrhardt R.A.
      • Foskolos A.
      • Giesy S.L.
      • Wesolowski S.R.
      • Krumm C.S.
      • Butler W.
      • Quirk S.
      • Waldron M.R.
      • Boisclair Y.R.
      Increased plasma leptin attenuates adaptive metabolism in early lactating dairy cows.
      was used to compare 2 assays in their ability to detect changes in plasma adiponectin between LP and early lactation (EL) and to assess the effects of leptin therapy in EL. Ten cows were fed unlimited amounts of TMR formulated for each physiological stage (1.5 Mcal of NEL and 140 g of CP per kg of DM in LP and 1.5 Mcal of NEL and 180 g of CP per kg of DM in EL). Plasma adiponectin was analyzed on 4 blood samples collected at 2-h intervals between 0800 and 1400 h in LP (d −29 ± 2, relative to parturition d 0) and again in EL (d +8). After completing blood sampling on d +8, cows were randomly assigned to receive a continuous intrajugular infusion of saline (saline) or human leptin (hLeptin, 61 μg/kg of BW/d; Eli Lilly and Company, Indianapolis, IN) for 96 consecutive hours. Plasma adiponectin was measured on 4 samples collected from each cow at 2-h intervals between 88 and 94 h of infusion.

      Energy Balance After Parturition

      An experiment described in
      • Block S.S.
      • Butler W.R.
      • Ehrhardt R.A.
      • Bell A.W.
      • Van Amburgh M.E.
      • Boisclair Y.R.
      Decreased concentration of plasma leptin in periparturient dairy cows is caused by negative energy balance.
      was used to evaluate the effect of positive energy balance after parturition on the recovery of plasma adiponectin. Between parturition and d +32 of lactation, cows were offered a low-energy TMR (1.52 Mcal of NEL and 189 g of CP per kg of DM) and milked thrice daily at 0900, 1600, and 2300 h (lactating, n = 7) or offered a high-energy TMR (1.70 Mcal of NEL and 188 g of CP per kg of DM) and never milked (nonlactating, n = 7). Lactating cows were fed ad libitum during the first week postpartum and thereafter limited to amounts consumed on d +7 (13.6 ± 1.2 kg/d); nonlactating cows were fed ad libitum at all times. Each cow was scored for body condition (thin = 1; fat = 5) on wk 1 and 4 by 2 independent individuals as previously described (
      • Block S.S.
      • Butler W.R.
      • Ehrhardt R.A.
      • Bell A.W.
      • Van Amburgh M.E.
      • Boisclair Y.R.
      Decreased concentration of plasma leptin in periparturient dairy cows is caused by negative energy balance.
      ). Plasma variables including adiponectin were analyzed on 4 blood samples collected from each cow every other day between d +5 and +11 and again between d +26 and +32.

      Effect of Insulin

      The study of
      • Leury B.J.
      • Baumgard L.H.
      • Block S.S.
      • Segoale N.
      • Ehrhardt R.A.
      • Rhoads R.P.
      • Bauman D.E.
      • Bell A.W.
      • Boisclair Y.R.
      Effect of insulin and growth hormone on plasma leptin in periparturient dairy cows.
      was used to assess the effect of chronic hyperinsulinemia during the transition period on plasma adiponectin. Six cows were fed unlimited amounts of appropriate TMR during LP (1.56 Mcal of NEL and 140 g of CP per kg of DM) and EL (1.58 Mcal of NEL and 198 g of CP per kg of DM). The cows were subjected to the hyperinsulinemic-euglycemic clamp procedure starting on d −31 ± 1.5 of LP and d +7 ± 1.6 of EL. The clamp procedure involved a 66-h period of basal blood sampling followed by infusion of bovine insulin at the rate of 1 µg/kg of BW per hour for 96 h in LP and 48 h in EL. During insulin infusion, plasma glucose levels were maintained to the concentration observed during the basal period by varying the rate of infusion of a 50% (wt/vol) dextrose solution (
      • Leury B.J.
      • Baumgard L.H.
      • Block S.S.
      • Segoale N.
      • Ehrhardt R.A.
      • Rhoads R.P.
      • Bauman D.E.
      • Bell A.W.
      • Boisclair Y.R.
      Effect of insulin and growth hormone on plasma leptin in periparturient dairy cows.
      ). Plasma adiponectin was determined on samples collected during the basal period (−66, −43, −30, and 0 h, relative to initiation of insulin infusion) and on 4 plasma samples collected from each cow at 4-h intervals between 36 and 48 h and 84 and 96 h of hyperinsulinemia during LP and between 36 and 48 h of hyperinsulinemia during EL.

      Effect of GH

      An experiment described in
      • Schoenberg K.M.
      • Giesy S.L.
      • Harvatine K.J.
      • Waldron M.R.
      • Cheng C.
      • Kharitonenkov A.
      • Boisclair Y.R.
      Plasma FGF21 is elevated by the intense lipid mobilization of lactation.
      was used to assess the effect of chronic GH treatment during the transition period on plasma adiponectin. In brief, cows were studied over a 5-d period either in LP (d −40 to −36, relative to parturition on d 0, n = 7) or EL (d +7 to +11, n = 7). Cows were offered unlimited amounts of TMR appropriate for each physiological stage (1.52 Mcal of NEL and 142 g of CP per kg of DM in LP and 1.72 Mcal of NEL and 179 g of CP per kg of DM in EL). On the first day of each period, basal blood samples were collected hourly over a 3-h period, followed by biopsy of adipose tissue from the tail-head subcutaneous depot. Immediately afterward, cows received an intrajugular bolus of GH (20 μg of recombinant bST per kg of BW; Monsanto Co., St. Louis, MO) and a second adipose tissue biopsy was taken 15 min later. The GH treatment was continued over the next 4 d via daily i.m. injections of 40 mg/d of recombinant bST. Blood samples were collected from each cow at hourly intervals between 1000 and 1300 h on the last day of treatment. Plasma IGF-I and adiponectin were analyzed on samples collected during basal sampling and on d 5 of GH treatment.

      Effect of Elevated Plasma Fatty Acids

      The study of
      • Caixeta L.S.
      • Giesy S.L.
      • Krumm C.S.
      • Perfield 2nd, J.W.
      • Butterfield A.
      • Schoenberg K.M.
      • Beitz D.C.
      • Boisclair Y.R.
      Effect of circulating glucagon and free fatty acids on hepatic FGF21 production in dairy cows.
      was used to assess the effects of increased plasma fatty acids on plasma adiponectin. Six nonpregnant, nonlactating dairy cows consumed a single TMR (1.54 Mcal of NEL and 143 g of CP per kg of DM) in amounts covering 121% of energy requirements according to the
      • NRC
      throughout the entire experiment. Cows were randomly assigned to two 3 × 3 Latin squares with experimental periods of 17 h separated by 3-d intervals. Each experimental period included basal blood sampling over 1 h followed by a 16-h period of treatment. Treatments consisted of various combinations of s.c. injections of saline or bovine glucagon (Eli Lilly and Company) and i.v. infusion of saline or 20% intralipid solution (Frasenius, Kabi, Deerfield, IL). These treatments were i.v. infusion and s.c. injections of saline (saline), i.v. infusion of intralipid and s.c. injection of saline (intralipid) and i.v. infusion of intralipid and s.c. injection of glucagon (intralipid + glucagon). Intralipid and saline solutions were infused at the rate of 100 mL/h for 16 consecutive hours and glucagon was administered at the dose of 5 mg at 0 and 8 h relative to the start of treatment. Blood samples were obtained from each cow during the basal period (−1 and −0.5 h relative to treatment at 0 h) and during treatment (0, +3, +7, +10, +13, and +16 h) analyzed for plasma fatty acids and adiponectin.

      Analysis of Metabolites and Hormones

      Plasma fatty acids were measured by the acyl-CoA synthetase/oxidase method described previously (
      • Block S.S.
      • Butler W.R.
      • Ehrhardt R.A.
      • Bell A.W.
      • Van Amburgh M.E.
      • Boisclair Y.R.
      Decreased concentration of plasma leptin in periparturient dairy cows is caused by negative energy balance.
      ). Plasma insulin and IGF-I were measured with double-antibody RIA as previously described (
      • Block S.S.
      • Butler W.R.
      • Ehrhardt R.A.
      • Bell A.W.
      • Van Amburgh M.E.
      • Boisclair Y.R.
      Decreased concentration of plasma leptin in periparturient dairy cows is caused by negative energy balance.
      ;
      • Leury B.J.
      • Baumgard L.H.
      • Block S.S.
      • Segoale N.
      • Ehrhardt R.A.
      • Rhoads R.P.
      • Bauman D.E.
      • Bell A.W.
      • Boisclair Y.R.
      Effect of insulin and growth hormone on plasma leptin in periparturient dairy cows.
      ;
      • Rhoads R.P.
      • Kim J.W.
      • Leury B.J.
      • Baumgard L.H.
      • Segoale N.
      • Frank S.J.
      • Bauman D.E.
      • Boisclair Y.R.
      Insulin increases the abundance of the growth hormone receptor in liver and adipose tissue of periparturient dairy cows.
      ). Plasma leptin was measured with a double-antibody RIA detecting only hLeptin (Linco Research Inc., St. Charles, MI) or bovine leptin (
      • Ehrhardt R.A.
      • Foskolos A.
      • Giesy S.L.
      • Wesolowski S.R.
      • Krumm C.S.
      • Butler W.
      • Quirk S.
      • Waldron M.R.
      • Boisclair Y.R.
      Increased plasma leptin attenuates adaptive metabolism in early lactating dairy cows.
      ). Effective leptin concentration in hLeptin-infused cows was calculated as the sum of bovine leptin plus human leptin corrected for its ability to activate the bovine leptin receptor (
      • Ehrhardt R.A.
      • Foskolos A.
      • Giesy S.L.
      • Wesolowski S.R.
      • Krumm C.S.
      • Butler W.
      • Quirk S.
      • Waldron M.R.
      • Boisclair Y.R.
      Increased plasma leptin attenuates adaptive metabolism in early lactating dairy cows.
      ). Inter-assay and intraassay coefficients of variation for these assays averaged less than 6 and 8%, respectively.
      Plasma adiponectin was measured by an indirect competitive ELISA designed to measure bovine adiponectin (
      • Mielenz M.
      • Mielenz B.
      • Singh S.P.
      • Kopp C.
      • Heinz J.
      • Häussler S.
      • Sauerwein H.
      Development, validation, and pilot application of a semiquantitative Western blot analysis and an ELISA for bovine adiponectin.
      ). The assay was performed as originally described, except for the following modifications. The calibration curve was prepared by a 4-fold serial dilution of the reference serum and ranged from 0.08 to 20 ng/mL. Other modifications included dilution of samples (1:20,000 instead of 1:80,000), volume of diluted samples and standards inputted in the assay (20 μL instead of 50 μL), and dilution of the primary antibody (1:5,000 instead of 1:2,000). The assay was developed with 100 μL of secondary antibody [donkey anti-rabbit horseradish peroxidase (Jackson ImmunoResearch #711–035–152, West Grove, PA); 1:15,000 dilution in assay buffer], and 100 μL of substrate solution [SureBlue TMB Microwell Peroxidase Substrate (KPL), Gaithersburg, MD]. The reaction was stopped by adding 100 μL of TMB stop solution (KPL) followed by reading of the optical density at 450 nm with a microplate reader. Inter-assay and intraassay coefficients of variation for all assays averaged less than 6.4 and 8%, respectively. Plasma adiponectin was also measured in a subset of samples by Western immunoblotting exactly as described recently (
      • Giesy S.L.
      • Yoon B.
      • Currie W.B.
      • Kim J.W.
      • Boisclair Y.R.
      Adiponectin deficit during the precarious glucose economy of early lactation in dairy cows.
      ). In brief, plasma samples (1.5 μL of 1:10 dilution for plasma) were electrophoresed under reducing conditions on 13% SDS PAGE gels and electroblotted onto nitrocellulose membranes (Protran, Schleicher & Schuell Bioscience, Keene, NH). The membranes were incubated with bovine adiponectin antiserum (1:1,000 dilution) and developed with a 1:20,000 dilution of IR Dye 800-nm goat anti-rabbit secondary antibody (LI-COR Biotechnology, Lincoln, NE). Signals were quantified with the LI-COR Odyssey infrared imaging system using the 800-nm channel.

      Western Immunoblotting of Adipose Tissue Extract

      Bovine adipose tissue was homogenized in 2 mL of lysis buffer [10 mM Tris, pH 7.6, 10 mL/L of Triton X-100, 1 mM EGTA, 150 mM NaCl, 1 mM Na3VO4, 1 mM Na pyrophosphate, 10 mM NaF, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 mg/L of aprotinin, and 10 mg/L of leupeptin]. Homogenates were clarified by centrifugation (10,000 × g for 20 min at 4°C). Protein concentrations of cellular extracts were determined using a bicinchoninic acid assay protein assay kit (Thermo Fisher, Waltham, MA). Fixed amounts (55 μg) of protein extract were separated on 10% polyacrylamide gels and transferred onto nitrocellulose membranes (Protran, Schleicher, and Schuell Bioscience, Dassel, Germany). Membranes were immersed in blocking solution (50 mM Tris, pH 7.4, 200 mM NaCl, 1 mL/L of Tween 20, 50 g/L of nonfat dried skim milk). Membranes were then immunodecorated overnight at 4°C with a 1:4,000 dilution of antibodies against signal transducer and activator of transcription-5 (STAT5; Cell Signaling, Danvers, MA) or a 1:1,000 dilution of antibodies against tyrosine phosphorylated Stat5 (pSTAT5; Cell Signaling). Signals were developed with a 1:5,000 dilution of goat anti-rabbit antibody (Thermo Fisher) in 5% nonfat dried skim milk and visualized by chemiluminescence exposure to film (Super Signal West Pico Chemiluminescent Substrate, Thermo Fisher). Signals were quantified by densitometry using the National Institutes of Health 1.6.3 software (National Institutes of Health, Bethesda, MD).

      Statistical Analysis

      Data were analyzed by a mixed model using the fit model procedure of JMP Pro 11.0 statistical software (SAS Institute Inc., Cary, NC). Data from the periparturient period study were analyzed by a mixed model accounting for physiological stage (LP or EL) as the fixed effect and cow as the random effect. For the leptin infusion portion of the experiment, data were analyzed with the fixed effect of treatment (control or hLeptin) and cow as the random effect with data obtained on d 8 used as a covariate. For the energy balance study, the mixed model accounted for treatment (lactating vs. nonlactating), time (wk 1 vs. 4), and their interaction as fixed effects and cow as the random effect. For the insulin and GH studies, the mixed model accounted for physiological stage (LP vs. EL), treatment (either basal vs. insulin or GH), and their interaction as fixed effects and cow as the random effect. For the plasma fatty acid study, the mixed model accounted for treatment (saline, intralipid, and intralipid + glucagon), time (0, 3, 7, 10, 13, and 16 h), and their interaction as fixed effects, and cow as the random effect. Values obtained during the basal period (average of −1 and −0.5 h samples) were used as a covariate. Correlations between plasma adiponectin and other variables were performed using the fit model procedure of JMP. Statistical significance was set at P < 0.05.

      RESULTS

      Comparison of Adiponectin Assays

      In previous work, we reported a 30% reduction in plasma adiponectin between d −35 and +7 relative to parturition on d 0 when measured by Western immunoblotting (
      • Giesy S.L.
      • Yoon B.
      • Currie W.B.
      • Kim J.W.
      • Boisclair Y.R.
      Adiponectin deficit during the precarious glucose economy of early lactation in dairy cows.
      ). This technique has limited applicability for the analysis of numerous samples across multiple studies. Accordingly, we used the transition cow study of
      • Ehrhardt R.A.
      • Foskolos A.
      • Giesy S.L.
      • Wesolowski S.R.
      • Krumm C.S.
      • Butler W.
      • Quirk S.
      • Waldron M.R.
      • Boisclair Y.R.
      Increased plasma leptin attenuates adaptive metabolism in early lactating dairy cows.
      to compare a recently developed bovine adiponectin ELISA to the Western immunoblotting assay. Energy indicators in that study varied as expected, with energy balance and plasma fatty acids averaging 12.1 Mcal of NEL per d and 108 μM in LP and −15.2 Mcal of NEL per d and 386 μM in EL. When measured by ELISA, plasma adiponectin fell from 51 to 36 μg/mL between d −29 of LP and d 8 of EL (P < 0.05). Moreover, both assays gave similar relative reductions in plasma adiponectin between LP and EL (Figure 1A). The ELISA assay was adopted for all subsequent analyses.
      Figure thumbnail gr1
      Figure 1Comparison of adiponectin assays and effect of increased plasma leptin in early lactation on plasma adiponectin. Multiparous dairy cows were studied in late pregnancy (LP) on d −29 ± 2 (relative to parturition on d 0) and again in early lactation (EL) on d +8. (A) Plasma samples were collected at 2-h intervals over a 6-h sampling window at both times and analyzed for adiponectin by Western immunoblotting or by a bovine adiponectin ELISA. The percent reduction in plasma adiponectin between LP and EL is shown. Each bar represents the mean ± SE of 10 cows. (B) Upon completion of the sampling period on d +8 of EL, dairy cows were randomly assigned to a constant intravenous infusion of saline (saline) or human leptin (hLeptin) for 96 consecutive hours. Plasma samples were collected between 88 and 94 h of infusion and analyzed for adiponectin concentration with a bovine adiponectin ELISA. Each bar represents the mean ± SE of 5 cows per treatment.

      Effect of Energy Balance Immediately After Parturition

      Next, we analyzed samples from cows milked thrice daily (lactating) or never milked after parturition (nonlactating) to ask whether eliminating the energy deficit of EL affected plasma adiponectin. Daily energy balance and the change in BCS over the 4 wk of the study were −16.8 Mcal of NEL and −0.6 units for lactating cows, and +12.1 Mcal of NEL and 0.1 unit for nonlactating cows (P < 0.001 for both variables). Plasma adiponectin increased by an average of 24% in both groups between wk 1 and 4 (Figure 2A, time, P < 0.001) and was 21% higher across time in nonlactating than lactating cows (treatment, P < 0.05). When assessed across treatments, no relation existed between the change in body condition over this period and the change in plasma adiponectin (Figure 2B). Modest relations were observed, however, between adiponectin and plasma indicators of energy status, with adiponectin increasing with leptin or insulin and decreasing with plasma fatty acids (Figure 2C and results not shown, P < 0.05 or less).
      Figure thumbnail gr2
      Figure 2Effect of energy balance after parturition on plasma adiponectin. (A) After parturition, multiparous dairy cows were milked thrice daily (lactating) or never milked (nonlactating). Plasma samples were collected between d +5 and +11 (wk 1) and again between d +26 and +33 (wk 4) and analyzed for adiponectin concentration with a bovine adiponectin ELISA. Each bar represents the mean ± SE of 7 cows per treatment. The significant effects of treatment and time are reported. (B) Relationship between the change in plasma adiponectin (Δ plasma adiponectin) and the change in body condition (Δ body condition) between wk 4 and 1 of the study. (C, D) Relationships between plasma adiponectin and plasma leptin or plasma fatty acids over the first 4 wk following parturition.

      Reversal of the Insulin and Leptin Deficits of Early Lactation

      The plasma concentrations of leptin and insulin fall over the last few days of pregnancy and reach a nadir after parturition (
      • Block S.S.
      • Butler W.R.
      • Ehrhardt R.A.
      • Bell A.W.
      • Van Amburgh M.E.
      • Boisclair Y.R.
      Decreased concentration of plasma leptin in periparturient dairy cows is caused by negative energy balance.
      ;
      • Rhoads R.P.
      • Kim J.W.
      • Leury B.J.
      • Baumgard L.H.
      • Segoale N.
      • Frank S.J.
      • Bauman D.E.
      • Boisclair Y.R.
      Insulin increases the abundance of the growth hormone receptor in liver and adipose tissue of periparturient dairy cows.
      ). To determine whether a fall in plasma leptin contributes to reduced adiponectin, cows received continuous i.v. infusions of saline or hLeptin between d 8 and 12 of lactation. Relative to saline infusion, the hLeptin infusion caused a 3.5-fold increase in the effective plasma leptin concentration (2.3 vs. 8 ng/mL, P < 0.001) and a 45% increase in plasma T3 (0.91 vs. 1.32 ng/mL, P < 0.01), but did not affect energy variables such as feed intake and milk energy output (
      • Ehrhardt R.A.
      • Foskolos A.
      • Giesy S.L.
      • Wesolowski S.R.
      • Krumm C.S.
      • Butler W.
      • Quirk S.
      • Waldron M.R.
      • Boisclair Y.R.
      Increased plasma leptin attenuates adaptive metabolism in early lactating dairy cows.
      ). Plasma adiponectin remained unaffected after 94 h of hLeptin treatment (Figure 1B).
      Next, we asked whether insulin had an effect on plasma adiponectin in either LP or EL. Dairy cows underwent a hyperinsulinemic-euglycemic clamp for 96 h in LP and for 48 h in EL. Plasma insulin increased within 4 h from 1.8 to 4.0 ng/mL in LP and from 0.7 to 2.5 ng/mL in EL, whereas the plasma glucose concentration remained within 10% of basal concentration at all times (
      • Leury B.J.
      • Baumgard L.H.
      • Block S.S.
      • Segoale N.
      • Ehrhardt R.A.
      • Rhoads R.P.
      • Bauman D.E.
      • Bell A.W.
      • Boisclair Y.R.
      Effect of insulin and growth hormone on plasma leptin in periparturient dairy cows.
      ). Plasma adiponectin fell between LP and EL (Figure 3, stage, P < 0.001) but was unaffected after 48 h of hyperinsulinemia in either LP or EL. Prolonging the period of hyperinsulinemia to 96 h during LP was also without effect on plasma adiponectin (basal vs. 96 h, 54 vs. 56 ng/mL).
      Figure thumbnail gr3
      Figure 3Effect of euglycemic-hyperinsulinemia in late pregnancy and early lactation on plasma adiponectin. Multiparous dairy cows during late pregnancy (LP; d −31 ± 1.5, relative to parturition on d 0) and early lactation (EL; +7 ± 1.6) were studied for 66 h under basal condition (basal) and for 48 h during euglycemic-hyperinsulinemia (insulin). Plasma samples collected during the basal period and between 36 and 48 h of euglycemic-hyperinsulinemia were analyzed for adiponectin concentration with a bovine adiponectin ELISA. Each bar represents the mean ± SE of 6 cows. The significant effect of physiological stage (stage) is reported.

      Increasing Plasma GH or Fatty Acids

      Plasma GH and fatty acids increase in periparturient dairy cows (
      • Block S.S.
      • Butler W.R.
      • Ehrhardt R.A.
      • Bell A.W.
      • Van Amburgh M.E.
      • Boisclair Y.R.
      Decreased concentration of plasma leptin in periparturient dairy cows is caused by negative energy balance.
      ;
      • Rhoads R.P.
      • Kim J.W.
      • Leury B.J.
      • Baumgard L.H.
      • Segoale N.
      • Frank S.J.
      • Bauman D.E.
      • Boisclair Y.R.
      Insulin increases the abundance of the growth hormone receptor in liver and adipose tissue of periparturient dairy cows.
      ). To assess the possible regulatory influence of GH on plasma adiponectin, LP or EL cows were treated with recombinant bST for 5 consecutive days. Plasma IGF-I was lower in EL than LP cows under basal condition (Figure 4A, stage, P < 0.001). After 5 d of treatment, plasma IGF-I was increased by GH treatment but to a lesser extent in EL than LP (stage × GH, P < 0.05). Adipose tissue, on the other hand, remained fully responsive to GH as shown by near identical GH-dependent STAT5 phosphorylation across physiological states (Figure 4B). Plasma adiponectin was again reduced in EL relative to LP (Figure 4C, stage, P < 0.05) but was unaffected by GH in either LP or EL.
      Figure thumbnail gr4
      Figure 4Effect of growth hormone in late pregnancy and early lactation on plasma adiponectin. Multiparous dairy cows were studied in late pregnancy (LP) between d −40 and −36 (relative to parturition on d 0) and in early lactation (EL) between d +7 and +11 over a 5-d period. On the first day of the study period, basal blood samples were collected hourly over a 3-h period. Adipose tissue was then collected before and 15 min after an intrajugular growth hormone (GH) bolus (20 μg of recombinant bST/kg of BW). On d 2 to 5, cows received daily injections of GH (40 mg of recombinant bST/d). (A) Plasma samples collected during the basal period (basal) and on d 5 of GH treatment (GH) were analyzed for plasma IGF-I. Each bar represents the mean ± SE of 7 cows per treatment and stage. The significant effects of physiological stage (stage), GH treatment, and their interaction are reported. (B) Left: Adipose tissue was obtained on d 1 of the study period immediately before (−) and 15 min after (+) an intrajugular bolus of recombinant bST (GH). Total cellular extracts were prepared and analyzed for tyrosine phosphorylated Stat5 (pStat5) and total Stat5 (Stat5) by Western immunoblotting. A representative sample is shown for each stage. Right: the GH-dependent pStat5 signal was normalized to total Stat5 signal and expressed relative to LP level with each bar representing the mean ± SE of 7 cows. (C) Plasma samples collected during the basal period (basal) and on d 5 of GH treatment (GH) were analyzed for adiponectin concentration with a bovine adiponectin ELISA. Each bar represents the mean ± SE of 7 cows per stage. The significant effect of physiological stage (stage) is reported.
      Finally, the possibility that plasma fatty acids repress plasma adiponectin was evaluated by i.v. infusion of a lipid emulsion in nonpregnant, nonlactating cows in the absence or presence of glucagon for 16 consecutive hours. Intralipid caused a 6-fold increase in plasma fatty acids within 3 h of infusion (saline vs. intralipid, 102 vs. 576 μM, P < 0.001), and this increase persisted for the remainder of the infusion; glucagon had no additional effect on plasma fatty acids (intralipid + glucagon, 613 μM). Plasma adiponectin remained unaffected throughout intralipid infusion irrespective of the presence or absence of glucagon (Figure 5).
      Figure thumbnail gr5
      Figure 5Effect of increased plasma fatty acids on plasma adiponectin. Nonpregnant, nonlactating dairy cows were treated over 16 h with combinations of i.v. infusion of saline or intralipid 20% and s.c. injections of saline or glucagon. Treatments were i.v. infusion and s.c. injections of saline (saline), i.v. infusion of intralipid and s.c. injections of saline (intralipid), or i.v. infusion of intralipid and s.c. injection of glucagon (intralipid + glucagon). Plasma samples were collected over the 16-h periods and analyzed for adiponectin concentration with a bovine adiponectin ELISA. Each point represents the mean ± SE of 6 cows.

      DISCUSSION

      Insulin resistance is a mechanism used by the modern dairy cow to promote glucose partitioning to the mammary gland at the expense of insulin-sensitive tissues such as skeletal muscle or adipose tissue (
      • Bell A.W.
      • Bauman D.E.
      Adaptations of glucose metabolism during pregnancy and lactation.
      ;
      • Boisclair Y.R.
      • Wesolowski S.R.
      • Kim J.W.
      • Ehrhardt R.A.
      ). Mechanisms regulating IR during EL in dairy cows are not well understood. In this context, we and others have shown that plasma adiponectin drops in parallel with increased IR in periparturient dairy cows and that this reduction occurs in the absence of a corresponding change in adiponectin mRNA in adipose tissue (
      • Lemor A.
      • Hosseini A.
      • Sauerwein H.
      • Mielenz M.
      Transition period-related changes in the abundance of the mRNAs of adiponectin and its receptors, of visfatin, and of fatty acid binding receptors in adipose tissue of high-yielding dairy cows.
      ;
      • Giesy S.L.
      • Yoon B.
      • Currie W.B.
      • Kim J.W.
      • Boisclair Y.R.
      Adiponectin deficit during the precarious glucose economy of early lactation in dairy cows.
      ;
      • Mielenz M.
      • Mielenz B.
      • Singh S.P.
      • Kopp C.
      • Heinz J.
      • Häussler S.
      • Sauerwein H.
      Development, validation, and pilot application of a semiquantitative Western blot analysis and an ELISA for bovine adiponectin.
      ;
      • Singh S.P.
      • Häussler S.
      • Gross J.J.
      • Schwarz F.J.
      • Bruckmaier R.M.
      • Sauerwein H.
      Short communication: circulating and milk adiponectin change differently during energy deficiency at different stages of lactation in dairy cows.
      ). Adiponectin is an insulin-sensitizing hormone (
      • Kadowaki T.
      • Yamauchi T.
      • Kubota N.
      • Hara K.
      • Ueki K.
      • Tobe K.
      Adiponectin and adiponectin receptors in insulin resistance, diabetes, and the metabolic syndrome.
      ;
      • Wang Z.V.
      • Scherer P.E.
      Adiponectin, the past two decades.
      ), and therefore, its reduction could contribute to the IR of periparturient dairy cows. Virtually no information is available on mechanisms regulating plasma adiponectin in periparturient dairy cows, prompting us to examine possible regulatory effects of metabolic and hormonal factors changing in a dynamic manner during this period.
      The profile of plasma adiponectin in multiparous periparturient dairy cows consists of 2 reciprocal patterns, namely a falling phase over the last 2 to 3 wk of pregnancy followed by a rising phase after parturition (
      • Giesy S.L.
      • Yoon B.
      • Currie W.B.
      • Kim J.W.
      • Boisclair Y.R.
      Adiponectin deficit during the precarious glucose economy of early lactation in dairy cows.
      ;
      • Singh S.P.
      • Häussler S.
      • Gross J.J.
      • Schwarz F.J.
      • Bruckmaier R.M.
      • Sauerwein H.
      Short communication: circulating and milk adiponectin change differently during energy deficiency at different stages of lactation in dairy cows.
      ). An obvious question is whether the placenta could account for the falling phase through reduced adiponectin production in LP and its expulsion at parturition. More recent studies performed on mid- and late-gestation human placenta, primary human cytotrophoblasts, and placental choriocarcinoma cell lines (Jeg-3, JAR, and BeWo) have ruled out the placenta as a source of adiponectin (
      • Haugen F.
      • Ranheim T.
      • Harsem N.K.
      • Lips E.
      • Staff A.C.
      • Drevon C.A.
      Increased plasma levels of adipokines in preeclampsia: relationship to placenta and adipose tissue gene expression.
      ;
      • Pinar H.
      • Basu S.
      • Hotmire K.
      • Laffineuse L.
      • Presley L.
      • Carpenter M.
      • Catalano P.M.
      • Hauguel-de Mouzon S.
      High molecular mass multimer complexes and vascular expression contribute to high adiponectin in the fetus.
      ;
      • McDonald E.A.
      • Wolfe M.W.
      Adiponectin attenuation of endocrine function within human term trophoblast cells.
      ). Moreover, adiponectin mRNA abundance was less than 0.5% of that of maternal adipose tissue at both d 50 and 135 of gestation in the sheep (Y. R. Boisclair, unpublished data). Given the close evolutionary relationship between sheep and cattle, it is unlikely that the bovine placenta is a source of adiponectin and a contributing factor to its falling plasma concentration in late pregnant dairy cows.
      In rodents and humans, loss of adiposity as well as NEB are associated with increasing plasma adiponectin (
      • Kadowaki T.
      • Yamauchi T.
      • Kubota N.
      • Hara K.
      • Ueki K.
      • Tobe K.
      Adiponectin and adiponectin receptors in insulin resistance, diabetes, and the metabolic syndrome.
      ). The relation between energy balance or adiposity and plasma adiponectin has been investigated in nonlactating, nonpregnant dairy cows and in cows from the last month of gestation through the first 12 wk of lactation (
      • Singh S.P.
      • Häussler S.
      • Gross J.J.
      • Schwarz F.J.
      • Bruckmaier R.M.
      • Sauerwein H.
      Short communication: circulating and milk adiponectin change differently during energy deficiency at different stages of lactation in dairy cows.
      ;
      • Locher L.
      • Häussler S.
      • Laubenthal L.
      • Singh S.P.
      • Winkler J.
      • Kinoshita A.
      • Kenéz Á.
      • Rehage J.
      • Huber K.
      • Sauerwein H.
      • Dänicke S.
      Effect of increasing body condition on key regulators of fat metabolism in subcutaneous adipose tissue depot and circulation of nonlactating dairy cows.
      ). The contribution of these factors to changes in plasma adiponectin around parturition remains unclear. Accordingly, we used dairy cows dried off immediately after parturition to test the possibility that these factors contribute to the recovery of plasma adiponectin after parturition. In particular, the nonlactating dairy cows completely avoided energy insufficiency after parturition and did not experience a loss of adiposity over the next 4 wk. Against expectations, nonlactating dairy cows not only maintained higher plasma adiponectin than lactating dairy cows but also recovered plasma adiponectin at similar rates as lactating dairy cows even though they failed to lose body condition. Measurable amounts of adiponectin are secreted in milk (
      • Singh S.P.
      • Häussler S.
      • Gross J.J.
      • Schwarz F.J.
      • Bruckmaier R.M.
      • Sauerwein H.
      Short communication: circulating and milk adiponectin change differently during energy deficiency at different stages of lactation in dairy cows.
      ), raising the possibility that the higher plasma adiponectin of nonlactating dairy cows relates to absence of adiponectin excretion in milk. However, the fraction of the steady-state plasma adiponectin pool appearing daily in milk is 5% or less (
      • Singh S.P.
      • Häussler S.
      • Gross J.J.
      • Schwarz F.J.
      • Bruckmaier R.M.
      • Sauerwein H.
      Short communication: circulating and milk adiponectin change differently during energy deficiency at different stages of lactation in dairy cows.
      ). Moreover, previous studies in mice and humans suggest a half-life in the 75 to 150 min range for plasma adiponectin (
      • Hoffstedt J.
      • Arvidsson E.
      • Sjölin E.
      • Wåhlén K.
      • Arner P.
      Adipose tissue adiponectin production and adiponectin serum concentration in human obesity and insulin resistance.
      ;
      • Halberg N.
      • Schraw T.D.
      • Wang Z.V.
      • Kim J.-Y.
      • Yi J.
      • Hamilton M.P.
      • Luby-Phelps K.
      • Scherer P.E.
      Systemic fate of the adipocyte-derived factor adiponectin.
      ), implying that this fraction would be even less if calculated on the basis of daily adiponectin production. Therefore, milk adiponectin secretion cannot account for the 21% difference in plasma adiponectin between lactating and nonlactating dairy cows. Finally, the positive effect of energy balance after parturition on plasma adiponectin is in contrast to the lack of any response when dairy cows experience severe energy insufficiency in mid-lactation (
      • Singh S.P.
      • Häussler S.
      • Gross J.J.
      • Schwarz F.J.
      • Bruckmaier R.M.
      • Sauerwein H.
      Short communication: circulating and milk adiponectin change differently during energy deficiency at different stages of lactation in dairy cows.
      ). Overall, our data suggest that the reduction in plasma adiponectin after parturition is driven in part by NEB and that adiponectin recovery after parturition occurs independent of loss of adiposity.
      The periparturient period is characterized by rapid reductions in plasma leptin and insulin, raising the possibility that either one or both of these hormones are positive regulators of adiponectin production. In apparent support for this model, plasma adiponectin increases after leptin therapy of leptin-deficient humans and mice (
      • Delporte M.-L.
      • El Mkadem S.A.
      • Quisquater M.
      • Brichard S.M.
      Leptin treatment markedly increased plasma adiponectin but barely decreased plasma resistin of ob/ob mice.
      ;
      • Licinio J.
      • Caglayan S.
      • Ozata M.
      • Yildiz B.O.
      • de Miranda P.B.
      • O'Kirwan F.
      • Whitby R.
      • Liang L.
      • Cohen P.
      • Bhasin S.
      • Krauss R.M.
      • Veldhuis J.D.
      • Wagner A.J.
      • DePaoli A.M.
      • McCann S.M.
      • Wong M.-L.
      Phenotypic effects of leptin replacement on morbid obesity, diabetes mellitus, hypogonadism, and behavior in leptin-deficient adults.
      ). However, we failed to detect increased plasma adiponectin in EL dairy cows, despite a 3.5-fold increase in plasma leptin. It is also important to mention that the positive effect of leptin therapy seen in leptin-deficient humans and mice is likely a consequence of substantial loss of adiposity rather than a direct leptin effect. In similar fashion to the lack of leptin effects, increasing plasma insulin in either LP or EL was also without any effects on plasma adiponectin. This result is not only inconsistent with a positive role for insulin in the cow but also discordant with results in humans and rodents showing that loss of function mutations of the insulin receptor increase plasma adiponectin, whereas euglycemic-hyperinsulinemia reduces plasma adiponectin (
      • Blüher M.
      • Michael M.D.
      • Peroni O.D.
      • Ueki K.
      • Carter N.
      • Kahn B.B.
      • Kahn C.R.
      Adipose tissue selective insulin receptor knockout protects against obesity and obesity-related glucose intolerance.
      ;
      • Semple R.K.
      • Soos M.A.
      • Luan J.
      • Mitchell C.S.
      • Wilson J.C.
      • Gurnell M.
      • Cochran E.K.
      • Gorden P.
      • Chatterjee V.K.K.
      • Wareham N.J.
      • O'Rahilly S.
      Elevated plasma adiponectin in humans with genetically defective insulin receptors.
      ;
      • Bobbert T.
      • Weicht J.
      • Mai K.
      • Möhlig M.
      • Pfeiffer A.F.H.
      • Spranger J.
      Acute hyperinsulinaemia and hyperlipidaemia modify circulating adiponectin and its oligomers.
      ). Interestingly, the effects of insulin in humans are not seen on high molecular weight adiponectin (
      • Bobbert T.
      • Weicht J.
      • Mai K.
      • Möhlig M.
      • Pfeiffer A.F.H.
      • Spranger J.
      Acute hyperinsulinaemia and hyperlipidaemia modify circulating adiponectin and its oligomers.
      ). Accordingly, it is possible that the lack of insulin effect in dairy cows relates to circulation of adiponectin predominantly in the high molecular weight form (
      • Giesy S.L.
      • Yoon B.
      • Currie W.B.
      • Kim J.W.
      • Boisclair Y.R.
      Adiponectin deficit during the precarious glucose economy of early lactation in dairy cows.
      ).
      Interestingly, hypothyroidism in human patients is associated with reduced plasma adiponectin, whereas hyperthyroidism in both humans and rodents has the opposite effect (
      • Yaturu S.
      • Prado S.
      • Grimes S.R.
      Changes in adipocyte hormones leptin, resistin, and adiponectin in thyroid dysfunction.
      ). Periparturient dairy cows are known to experience deficits in the plasma concentration of both thyroid hormones and IGF-I (
      • Reist M.
      • Erdin D.
      • von Euw D.
      • Tschuemperlin K.
      • Leuenberger H.
      • Delavaud C.
      • Chilliard Y.
      • Hammon H.M.
      • Kuenzi N.
      • Blum J.W.
      Concentrate feeding strategy in lactating dairy cows: metabolic and endocrine changes with emphasis on leptin.
      ;
      • Ehrhardt R.A.
      • Foskolos A.
      • Giesy S.L.
      • Wesolowski S.R.
      • Krumm C.S.
      • Butler W.
      • Quirk S.
      • Waldron M.R.
      • Boisclair Y.R.
      Increased plasma leptin attenuates adaptive metabolism in early lactating dairy cows.
      ). However, our work shows that plasma adiponectin remained unaffected by correction of the thyroid hormone deficit during the leptin infusion (
      • Ehrhardt R.A.
      • Foskolos A.
      • Giesy S.L.
      • Wesolowski S.R.
      • Krumm C.S.
      • Butler W.
      • Quirk S.
      • Waldron M.R.
      • Boisclair Y.R.
      Increased plasma leptin attenuates adaptive metabolism in early lactating dairy cows.
      ) and correction of the IGF-I deficit during the euglycemic-hyperinsulinemia clamp (
      • Rhoads R.P.
      • Kim J.W.
      • Leury B.J.
      • Baumgard L.H.
      • Segoale N.
      • Frank S.J.
      • Bauman D.E.
      • Boisclair Y.R.
      Insulin increases the abundance of the growth hormone receptor in liver and adipose tissue of periparturient dairy cows.
      ).
      Plasma GH and fatty acids have been implicated as drivers of IR and both are increased in EL dairy cows (
      • Rhoads R.P.
      • Kim J.W.
      • Leury B.J.
      • Baumgard L.H.
      • Segoale N.
      • Frank S.J.
      • Bauman D.E.
      • Boisclair Y.R.
      Insulin increases the abundance of the growth hormone receptor in liver and adipose tissue of periparturient dairy cows.
      ;
      • Boisclair Y.R.
      • Wesolowski S.R.
      • Kim J.W.
      • Ehrhardt R.A.
      ). In the case of both mice and humans, existing data suggest the possibility that adiponectin mediates the effect of GH on IR. Thus, GH receptor knockout mice have elevated plasma adiponectin and insulin sensitivity despite increased fatness (
      • Lubbers E.R.
      • List E.O.
      • Jara A.
      • Sackman-Sala L.
      • Cordoba-Chacon J.
      • Gahete M.D.
      • Kineman R.D.
      • Boparai R.
      • Bartke A.
      • Kopchick J.J.
      • Berryman D.E.
      Adiponectin in mice with altered GH action: links to insulin sensitivity and longevity?.
      ). Similarly, increased insulin sensitivity and plasma adiponectin coexist in patients lacking a functional GH receptor (Laron dwarf) and both are reduced in patients with excessive GH secretion (
      • Lam K.S.-L.
      • Xu A.
      • Tan K.C.-B.
      • Wong L.-C.
      • Tiu S.-C.
      • Tam S.
      Serum adiponectin is reduced in acromegaly and normalized after correction of growth hormone excess.
      ;
      • Kanety H.
      • Hemi R.
      • Ginsberg S.
      • Pariente C.
      • Yissachar E.
      • Barhod E.
      • Funahashi T.
      • Laron Z.
      Total and high molecular weight adiponectin are elevated in patients with Laron syndrome despite marked obesity.
      ). Therefore, we tested the possibility that GH inhibits adiponectin production by treating LP and EL dairy cows with exogenous GH. As previously shown, plasma IGF-I was lower in EL than LP, and GH increased plasma IGF-I to a larger extent in LP than EL (
      • Block S.S.
      • Butler W.R.
      • Ehrhardt R.A.
      • Bell A.W.
      • Van Amburgh M.E.
      • Boisclair Y.R.
      Decreased concentration of plasma leptin in periparturient dairy cows is caused by negative energy balance.
      ;
      • Rhoads R.P.
      • Kim J.W.
      • Leury B.J.
      • Baumgard L.H.
      • Segoale N.
      • Frank S.J.
      • Bauman D.E.
      • Boisclair Y.R.
      Insulin increases the abundance of the growth hormone receptor in liver and adipose tissue of periparturient dairy cows.
      ). The attenuated increase in plasma IGF-I reflects reduced hepatic GH responsiveness as a result of loss of GH receptor expression in EL (
      • Wook Kim J.
      • Rhoads R.P.
      • Block S.S.
      • Overton T.R.
      • Frank S.J.
      • Boisclair Y.R.
      Dairy cows experience selective reduction of the hepatic growth hormone receptor during the periparturient period.
      ). On the other hand, our data show that adipose tissue remains GH-responsive in both LP and EL as demonstrated by equally robust GH-dependent STAT5 activation across physiological states. Despite this, GH treatment for 5 consecutive days had no effect on plasma adiponectin irrespective of physiological state.
      Plasma fatty acids also contribute to IR (
      • Pires J.A.A.
      • Souza A.H.
      • Grummer R.R.
      Induction of hyperlipidemia by intravenous infusion of tallow emulsion causes insulin resistance in Holstein cows.
      ) particularly when their levels are excessively elevated in EL as a consequence of overnutrition, obesity, or both in LP (
      • Holtenius K.
      • Agenäs S.
      • Delavaud C.
      • Chilliard Y.
      Effects of feeding intensity during the dry period. 2. metabolic and hormonal responses.
      ;
      • Janovick N.A.
      • Boisclair Y.R.
      • Drackley J.K.
      Prepartum dietary energy intake affects metabolism and health during the periparturient period in primiparous and multiparous Holstein cows.
      ). Plasma fatty acids promote IR by promoting synthesis of lipid intermediates such as ceramide species and diacylglycerol and by activating the production of pro-inflammatory cytokines such as tumor necrosis factor α (
      • Shi H.
      • Kokoeva M.V.
      • Inouye K.
      • Tzameli I.
      • Yin H.
      • Flier J.S.
      TLR4 links innate immunity and fatty acid-induced insulin resistance.
      ;
      • Sadri H.
      • Bruckmaier R.M.
      • Rahmani H.R.
      • Ghorbani G.R.
      • Morel I.
      • van Dorland H.A.
      Gene expression of tumour necrosis factor and insulin signalling-related factors in subcutaneous adipose tissue during the dry period and in early lactation in dairy cows.
      ;
      • Rico J.E.
      • Bandaru V.V.R.
      • Dorskind J.M.
      • Haughey N.J.
      • McFadden J.W.
      Plasma ceramides are elevated in overweight Holstein dairy cows experiencing greater lipolysis and insulin resistance during the transition from late pregnancy to early lactation.
      ). Interestingly, intralipid, ceramide, and tumor necrosis factor α have been shown to repress adiponectin production in vivo and in vitro (
      • Lim J.-Y.
      • Kim W.H.
      • Park S.I.
      GO6976 prevents TNF-alpha-induced suppression of adiponectin expression in 3T3–L1 adipocytes: Putative involvement of protein kinase C.
      ;
      • Bobbert T.
      • Weicht J.
      • Mai K.
      • Möhlig M.
      • Pfeiffer A.F.H.
      • Spranger J.
      Acute hyperinsulinaemia and hyperlipidaemia modify circulating adiponectin and its oligomers.
      ;
      • Correnti J.M.
      • Juskeviciute E.
      • Swarup A.
      • Hoek J.B.
      Pharmacological ceramide reduction alleviates alcohol-induced steatosis and hepatomegaly in adiponectin knockout mice.
      ). Despite these data implicating plasma fatty acids as a repressor of adiponectin production, our data show that a 6-fold increase in plasma fatty acids during intralipid infusion had no effect on plasma adiponectin. It remains possible that the 13-h period of exposure to elevated plasma fatty acids was too short to elicit a reduction in plasma adiponectin.
      In summary, plasma adiponectin in transition dairy cows is remarkably unresponsive to metabolic and hormonal factors changing in a dynamic manner around parturition period and shown to regulate circulating levels in other species and contexts. Future efforts should focus on the role of other important hormones (i.e., FGF21, resistin, etc.) as well as on depot-specific factors (subcutaneous vs. visceral or bone marrow adipocytes).

      ACKNOWLEDGMENTS

      This material is based on work supported by the National Institute of Food and Agriculture, USDA (Washington, DC) , under award number 2014-67015-21592 and Hatch/Multistate project under 1000962 .

      REFERENCES

        • Bell A.W.
        • Bauman D.E.
        Adaptations of glucose metabolism during pregnancy and lactation.
        J. Mammary Gland Biol. Neoplasia. 1997; 2 (10882310): 265-278
        • Block S.S.
        • Butler W.R.
        • Ehrhardt R.A.
        • Bell A.W.
        • Van Amburgh M.E.
        • Boisclair Y.R.
        Decreased concentration of plasma leptin in periparturient dairy cows is caused by negative energy balance.
        J. Endocrinol. 2001; 171 (11691654): 339-348
        • Blüher M.
        • Michael M.D.
        • Peroni O.D.
        • Ueki K.
        • Carter N.
        • Kahn B.B.
        • Kahn C.R.
        Adipose tissue selective insulin receptor knockout protects against obesity and obesity-related glucose intolerance.
        Dev. Cell. 2002; 3 (12110165): 25-38
        • Bobbert T.
        • Weicht J.
        • Mai K.
        • Möhlig M.
        • Pfeiffer A.F.H.
        • Spranger J.
        Acute hyperinsulinaemia and hyperlipidaemia modify circulating adiponectin and its oligomers.
        Clin. Endocrinol. (Oxf.). 2009; 71 (19751297): 507-511
        • Boisclair Y.R.
        • Wesolowski S.R.
        • Kim J.W.
        • Ehrhardt R.A.
        Sejrsen K. Hveplund T. Nielsen M.O. Role of growth hormone and leptin in the periparturient dairy cow. Wageningen Academic Publishers, Wageningen, the Netherlands2006
        • Caixeta L.S.
        • Giesy S.L.
        • Krumm C.S.
        • Perfield 2nd, J.W.
        • Butterfield A.
        • Schoenberg K.M.
        • Beitz D.C.
        • Boisclair Y.R.
        Effect of circulating glucagon and free fatty acids on hepatic FGF21 production in dairy cows.
        Am. J. Physiol. Regul. Integr. Comp. Physiol. 2017; (In press.)
        • Correnti J.M.
        • Juskeviciute E.
        • Swarup A.
        • Hoek J.B.
        Pharmacological ceramide reduction alleviates alcohol-induced steatosis and hepatomegaly in adiponectin knockout mice.
        Am. J. Physiol. Gastrointest. Liver Physiol. 2014; 306 (24742988): G959-G973
        • Delporte M.-L.
        • El Mkadem S.A.
        • Quisquater M.
        • Brichard S.M.
        Leptin treatment markedly increased plasma adiponectin but barely decreased plasma resistin of ob/ob mice.
        Am. J. Physiol. Endocrinol. Metab. 2004; 287 (15126241): E446-E453
        • Ehrhardt R.A.
        • Foskolos A.
        • Giesy S.L.
        • Wesolowski S.R.
        • Krumm C.S.
        • Butler W.
        • Quirk S.
        • Waldron M.R.
        • Boisclair Y.R.
        Increased plasma leptin attenuates adaptive metabolism in early lactating dairy cows.
        J. Endocrinol. 2016; (26957637)
        • Giesy S.L.
        • Yoon B.
        • Currie W.B.
        • Kim J.W.
        • Boisclair Y.R.
        Adiponectin deficit during the precarious glucose economy of early lactation in dairy cows.
        Endocrinology. 2012; 153 (23077076): 5834-5844
        • Halberg N.
        • Schraw T.D.
        • Wang Z.V.
        • Kim J.-Y.
        • Yi J.
        • Hamilton M.P.
        • Luby-Phelps K.
        • Scherer P.E.
        Systemic fate of the adipocyte-derived factor adiponectin.
        Diabetes. 2009; 58 (19581422): 1961-1970
        • Haugen F.
        • Ranheim T.
        • Harsem N.K.
        • Lips E.
        • Staff A.C.
        • Drevon C.A.
        Increased plasma levels of adipokines in preeclampsia: relationship to placenta and adipose tissue gene expression.
        Am. J. Physiol. Endocrinol. Metab. 2006; 290 (16144822): E326-E333
        • Hoffstedt J.
        • Arvidsson E.
        • Sjölin E.
        • Wåhlén K.
        • Arner P.
        Adipose tissue adiponectin production and adiponectin serum concentration in human obesity and insulin resistance.
        J. Clin. Endocrinol. Metab. 2004; 89 (15001639): 1391-1396
        • Holtenius K.
        • Agenäs S.
        • Delavaud C.
        • Chilliard Y.
        Effects of feeding intensity during the dry period. 2. metabolic and hormonal responses.
        J. Dairy Sci. 2003; 86 (12703625): 883-891
        • Hotta K.
        • Funahashi T.
        • Bodkin N.L.
        • Ortmeyer H.K.
        • Arita Y.
        • Hansen B.C.
        • Matsuzawa Y.
        Circulating concentrations of the adipocyte protein adiponectin are decreased in parallel with reduced insulin sensitivity during the progression to type 2 diabetes in rhesus monkeys.
        Diabetes. 2001; 50 (11334417): 1126-1133
        • Janovick N.A.
        • Boisclair Y.R.
        • Drackley J.K.
        Prepartum dietary energy intake affects metabolism and health during the periparturient period in primiparous and multiparous Holstein cows.
        J. Dairy Sci. 2011; 94 (21338804): 1385-1400
        • Kadowaki T.
        • Yamauchi T.
        • Kubota N.
        • Hara K.
        • Ueki K.
        • Tobe K.
        Adiponectin and adiponectin receptors in insulin resistance, diabetes, and the metabolic syndrome.
        J. Clin. Invest. 2006; 116 (16823476): 1784-1792
        • Kanety H.
        • Hemi R.
        • Ginsberg S.
        • Pariente C.
        • Yissachar E.
        • Barhod E.
        • Funahashi T.
        • Laron Z.
        Total and high molecular weight adiponectin are elevated in patients with Laron syndrome despite marked obesity.
        Eur. J. Endocrinol. 2009; 161 (19755405): 837-844
        • Kim J.-Y.
        • van de Wall E.
        • Laplante M.
        • Azzara A.
        • Trujillo M.E.
        • Hofmann S.M.
        • Schraw T.
        • Durand J.L.
        • Li H.
        • Li G.
        • Jelicks L.A.
        • Mehler M.F.
        • Hui D.Y.
        • Deshaies Y.
        • Shulman G.I.
        • Schwartz G.J.
        • Scherer P.E.
        Obesity-associated improvements in metabolic profile through expansion of adipose tissue.
        J. Clin. Invest. 2007; 117 (17717599): 2621-2637
        • Lam K.S.-L.
        • Xu A.
        • Tan K.C.-B.
        • Wong L.-C.
        • Tiu S.-C.
        • Tam S.
        Serum adiponectin is reduced in acromegaly and normalized after correction of growth hormone excess.
        J. Clin. Endocrinol. Metab. 2004; 89 (15531496): 5448-5453
        • Lemor A.
        • Hosseini A.
        • Sauerwein H.
        • Mielenz M.
        Transition period-related changes in the abundance of the mRNAs of adiponectin and its receptors, of visfatin, and of fatty acid binding receptors in adipose tissue of high-yielding dairy cows.
        Domest. Anim. Endocrinol. 2009; 37 (19345551): 37-44
        • Leury B.J.
        • Baumgard L.H.
        • Block S.S.
        • Segoale N.
        • Ehrhardt R.A.
        • Rhoads R.P.
        • Bauman D.E.
        • Bell A.W.
        • Boisclair Y.R.
        Effect of insulin and growth hormone on plasma leptin in periparturient dairy cows.
        Am. J. Physiol. Regul. Integr. Comp. Physiol. 2003; 285 (12881203): R1107-R1115
        • Licinio J.
        • Caglayan S.
        • Ozata M.
        • Yildiz B.O.
        • de Miranda P.B.
        • O'Kirwan F.
        • Whitby R.
        • Liang L.
        • Cohen P.
        • Bhasin S.
        • Krauss R.M.
        • Veldhuis J.D.
        • Wagner A.J.
        • DePaoli A.M.
        • McCann S.M.
        • Wong M.-L.
        Phenotypic effects of leptin replacement on morbid obesity, diabetes mellitus, hypogonadism, and behavior in leptin-deficient adults.
        Proc. Natl. Acad. Sci. USA. 2004; 101 (15070752): 4531-4536
        • Lim J.-Y.
        • Kim W.H.
        • Park S.I.
        GO6976 prevents TNF-alpha-induced suppression of adiponectin expression in 3T3–L1 adipocytes: Putative involvement of protein kinase C.
        FEBS Lett. 2008; 582 (18804108): 3473-3478
        • Locher L.
        • Häussler S.
        • Laubenthal L.
        • Singh S.P.
        • Winkler J.
        • Kinoshita A.
        • Kenéz Á.
        • Rehage J.
        • Huber K.
        • Sauerwein H.
        • Dänicke S.
        Effect of increasing body condition on key regulators of fat metabolism in subcutaneous adipose tissue depot and circulation of nonlactating dairy cows.
        J. Dairy Sci. 2015; 98 (25497790): 1057-1068
        • Lubbers E.R.
        • List E.O.
        • Jara A.
        • Sackman-Sala L.
        • Cordoba-Chacon J.
        • Gahete M.D.
        • Kineman R.D.
        • Boparai R.
        • Bartke A.
        • Kopchick J.J.
        • Berryman D.E.
        Adiponectin in mice with altered GH action: links to insulin sensitivity and longevity?.
        J. Endocrinol. 2013; 216 (23261955): 363-374
        • McDonald E.A.
        • Wolfe M.W.
        Adiponectin attenuation of endocrine function within human term trophoblast cells.
        Endocrinology. 2009; 150 (19520781): 4358-4365
        • Mielenz M.
        • Mielenz B.
        • Singh S.P.
        • Kopp C.
        • Heinz J.
        • Häussler S.
        • Sauerwein H.
        Development, validation, and pilot application of a semiquantitative Western blot analysis and an ELISA for bovine adiponectin.
        Domest. Anim. Endocrinol. 2013; 44 (23291015): 121-130
        • NRC
        Nutrient Requirements of Dairy Cattle. 7th rev. ed. Natl. Acad. Press, Washington, DC2001
        • Pinar H.
        • Basu S.
        • Hotmire K.
        • Laffineuse L.
        • Presley L.
        • Carpenter M.
        • Catalano P.M.
        • Hauguel-de Mouzon S.
        High molecular mass multimer complexes and vascular expression contribute to high adiponectin in the fetus.
        J. Clin. Endocrinol. Metab. 2008; 93 (18445668): 2885-2890
        • Pires J.A.A.
        • Souza A.H.
        • Grummer R.R.
        Induction of hyperlipidemia by intravenous infusion of tallow emulsion causes insulin resistance in Holstein cows.
        J. Dairy Sci. 2007; 90 (17517713): 2735-2744
        • Reist M.
        • Erdin D.
        • von Euw D.
        • Tschuemperlin K.
        • Leuenberger H.
        • Delavaud C.
        • Chilliard Y.
        • Hammon H.M.
        • Kuenzi N.
        • Blum J.W.
        Concentrate feeding strategy in lactating dairy cows: metabolic and endocrine changes with emphasis on leptin.
        J. Dairy Sci. 2003; 86 (12778580): 1690-1706
        • Rhoads R.P.
        • Kim J.W.
        • Leury B.J.
        • Baumgard L.H.
        • Segoale N.
        • Frank S.J.
        • Bauman D.E.
        • Boisclair Y.R.
        Insulin increases the abundance of the growth hormone receptor in liver and adipose tissue of periparturient dairy cows.
        J. Nutr. 2004; 134 (15113939): 1020-1027
        • Rico J.E.
        • Bandaru V.V.R.
        • Dorskind J.M.
        • Haughey N.J.
        • McFadden J.W.
        Plasma ceramides are elevated in overweight Holstein dairy cows experiencing greater lipolysis and insulin resistance during the transition from late pregnancy to early lactation.
        J. Dairy Sci. 2015; 98 (26342987): 7757-7770
        • Sadri H.
        • Bruckmaier R.M.
        • Rahmani H.R.
        • Ghorbani G.R.
        • Morel I.
        • van Dorland H.A.
        Gene expression of tumour necrosis factor and insulin signalling-related factors in subcutaneous adipose tissue during the dry period and in early lactation in dairy cows.
        J. Anim. Physiol. Anim. Nutr. (Berl.). 2010; 94 (20579185): e194-e202
        • Schoenberg K.M.
        • Giesy S.L.
        • Harvatine K.J.
        • Waldron M.R.
        • Cheng C.
        • Kharitonenkov A.
        • Boisclair Y.R.
        Plasma FGF21 is elevated by the intense lipid mobilization of lactation.
        Endocrinology. 2011; 152 (21990311): 4652-4661
        • Semple R.K.
        • Soos M.A.
        • Luan J.
        • Mitchell C.S.
        • Wilson J.C.
        • Gurnell M.
        • Cochran E.K.
        • Gorden P.
        • Chatterjee V.K.K.
        • Wareham N.J.
        • O'Rahilly S.
        Elevated plasma adiponectin in humans with genetically defective insulin receptors.
        J. Clin. Endocrinol. Metab. 2006; 91 (16705075): 3219-3223
        • Shi H.
        • Kokoeva M.V.
        • Inouye K.
        • Tzameli I.
        • Yin H.
        • Flier J.S.
        TLR4 links innate immunity and fatty acid-induced insulin resistance.
        J. Clin. Invest. 2006; 116 (17053832): 3015-3025
        • Singh S.P.
        • Häussler S.
        • Gross J.J.
        • Schwarz F.J.
        • Bruckmaier R.M.
        • Sauerwein H.
        Short communication: circulating and milk adiponectin change differently during energy deficiency at different stages of lactation in dairy cows.
        J. Dairy Sci. 2014; 97 (24472130): 1535-1542
        • Venn-Watson S.
        • Smith C.R.
        • Stevenson S.
        • Parry C.
        • Daniels R.
        • Jensen E.
        • Cendejas V.
        • Balmer B.
        • Janech M.
        • Neely B.A.
        • Wells R.
        Blood-based indicators of insulin resistance and metabolic syndrome in bottlenose dolphins (Tursiops truncatus).
        Front. Endocrinol. (Lausanne). 2013; 4 (24130551): 136
        • Wang Y.
        • Lam K.S.L.
        • Yau M.
        • Xu A.
        Post-translational modifications of adiponectin: mechanisms and functional implications.
        Biochem. J. 2008; 409 (18177270): 623-633
        • Wang Z.V.
        • Scherer P.E.
        Adiponectin, the past two decades.
        J. Mol. Cell Biol. 2016; 8 (26993047): 93-100
        • Wook Kim J.
        • Rhoads R.P.
        • Block S.S.
        • Overton T.R.
        • Frank S.J.
        • Boisclair Y.R.
        Dairy cows experience selective reduction of the hepatic growth hormone receptor during the periparturient period.
        J. Endocrinol. 2004; 181 (15128276): 281-290
        • Yaturu S.
        • Prado S.
        • Grimes S.R.
        Changes in adipocyte hormones leptin, resistin, and adiponectin in thyroid dysfunction.
        J. Cell. Biochem. 2004; 93 (15372626): 491-496