Inflammatory tone in liver and adipose tissue in dairy cows experiencing a healthy transition from late pregnancy to early lactation.

The transition from late pregnancy (LP) to early lactation (EL) in dairy cows is characterized by a major reorganization of the metabolic activities of liver and adipose tissue in support of milk synthesis. This reorganization has been attributed in large part to variation in the plasma concentration and actions of growth hormone, insulin, and other metabolic hormones. A role for the immune system has also been suggested by a near-universal rise in circulating levels of liver-derived acute-phase proteins (APP) in early lactating cows. However, less attention has been devoted to the possibility that resident macrophages of liver and adipose tissue adopt a proinflammatory state (referred herein as inflammatory tone) in parallel with the rise in plasma APP. We addressed this question by measuring the expression of genes expressed predominantly in the resident macrophage population of liver and adipose tissue and indicative of a proinflammatory (tumor necrosis factor α, IL-6, IL-12, resistin, and cluster of differentiation 80 [CD80]) or anti-inflammatory state (IL-10 and chitinase-3-like protein 1 [CHI3L1]). In a first group of cows, none of these inflammatory gene markers were regulated in liver between LP on d -29 (relative to parturition) and on d 8 of EL despite 1.7 to 5.6-fold upregulation in the expression of the APP (haptoglobin, serum amyloid α, and orosomucoid 1). In a second group of healthy cows, expression of the inflammatory gene markers did not differ between livers with low (<5.3%) or high (>11.5%) triglyceride content on d 7 of EL. In adipose tissue, a modest increase in inflammatory tone was suggested between LP and EL by increased CD80 expression and decreased CHI3L1 expression in EL. To assess the possibility that inflammatory tone would be more prominent if assayed in a cell compartment enriched with macrophages, adipose tissue was obtained in LP on d -28 and in EL on d +10 from cows experiencing a healthy transition period and fractionated into its adipocyte and stromal vascular cell (SVC) compartments. Expression of inflammatory gene markers was higher in SVC than adipocytes but remained unregulated in SVC between LP and EL. Overall, these results suggest little change in the inflammatory tone of resident macrophages in liver and adipose tissue of healthy transition dairy cows and do not support a role for the local immune system in the reorganization of metabolism in these tissues at the onset of lactation.

The acute phase protein orosomucoid 1 is upregulated in early lactation but does not trigger appetite-suppressing STAT3 signaling via the leptin receptor.

Dairy cows consume inadequate amounts of feed in early lactation and during conditions and diseases such as excessive fatness, heat stress, and infectious diseases. Affected cows often experience increases in plasma concentrations of acute phase proteins consistent with the negative effect of inflammation on appetite. The acute phase protein orosomucoid 1 (ORM1), also known as alpha-1-acid glycoprotein, was recently reported to reduce appetite in the mouse through its ability to bind the full-length leptin receptor (Ob-Rb) and activate appetite-suppressing signal transducer and activator of transcription 3 (STAT3) signaling. These observations raise the possibility that ORM1 exerts appetite-suppressing effects in dairy cattle during periods of increased inflammatory tone. The applicability of this model was assessed in 2 ways. First, we asked whether ORM1 is regulated during periods of inadequate appetite such as the transition from late pregnancy to early lactation and periods of increased inflammatory tone. Plasma ORM1 was invariant in late pregnancy but increased 2.5-fold between parturition and d 7 of lactation. Gene expression studies showed that liver was the major source of this elevation with little contribution by adipose tissue or mammary gland. Additional studies showed that plasma ORM1 was not increased further by excessive fatness or by reproductive dysfunction in early lactation and was completely unresponsive to inflammatory stimuli such as heat stress or intravascular administration of the endotoxin lipopolysaccharide during established lactation. Second, we tested the ability of ORM1 to trigger STAT3 signaling through Ob-Rb using Chinese hamster ovary K1 (CHO-K1) cells transfected with a STAT3 expression plasmid. In this configuration, CHO-K1 cells did not express Ob-Rb and were incapable of leptin-induced STAT3 phosphorylation. Leptin responsiveness was conferred by co-transfecting with bovine Ob-Rb, with leptin causing increases of 5.7-fold in STAT3 phosphorylation and 2.1-fold in the expression of the STAT3-dependent gene, SOCS3. In contrast, neither bovine or human ORM1 triggered STAT3 phosphorylation irrespective of dose and period of incubation tested. In summary, bovine ORM1 is not increased during periods of increased inflammatory tone except in early lactation and is incapable of Ob-Rb-dependent STAT3 signaling. Overall, these data are inconsistent with ORM1 mediating the appetite-suppressing effects of inflammation in cattle through Ob-Rb.

Fibroblast growth factor-21 (FGF21) administration to early-lactating dairy cows. I. Effects on signaling and indices of insulin action.

Modern dairy cows rely on hormonally driven mechanisms to coordinate the metabolic adaptations needed to meet the energy and nutrient deficits of early lactation. In the case of glucose, dairy cows cope with its scarcity during early lactation via reduced plasma concentrations of insulin and the insulin sensitizing hormone adiponectin and increased insulin resistance. Reduced insulin action promotes diversion of available glucose to the mammary gland but increases susceptibility to diseases if excessive. In earlier work, we reported that the insulin sensitizing hormone fibroblast growth factor-21 (FGF21) is increased in periparturient dairy cows and identified liver and adipose tissue as possible targets. These observations raised the possibility that FGF21 acts directly on these tissues to limit the insulin resistance of early lactation. To test this hypothesis, dairy cows were randomly assigned on d 12.6 ± 2.2 (± standard error) of lactation to receive either excipient (n = 6) or recombinant human FGF21 (n = 7), first as an FGF21 bolus of 3 mg/kg of body weight, followed 2 d later by a constant i.v. infusion of FGF21 at the rate of 6.3 mg/kg of metabolic body weight for 9 consecutive days. Biopsies of liver and adipose tissue were collected during the bolus phase of the experiment and used to analyze FGF21 signaling by Western blotting and expression of its receptor components by quantitative PCR. Bolus FGF21 administration caused a 4-fold increase in p44/42 MAPK (ERK1/2) activation in adipose tissue but had no effect on AKT and signal transducer and activator of transcription-3 (STAT3) signaling. The liver expressed negligible levels of the preferred FGF21 receptor FGFR1c and failed to mount any FGF21 signaling response. The FGF21 administered as a bolus had no effect on plasma glucose or insulin and did not stimulate an acute release of adiponectin from adipose tissue. Similarly, FGF21 infusion had no effect on plasma levels of glucose or insulin measured over the 9-d infusion or on glucose disposal during an i.v. glucose tolerance test performed on d 8 of infusion. Finally, the chronic FGF21 infusion had no effect on indices of adiponectin production, including plasma adiponectin and adipose tissue mRNA abundance of adiponectin and the endoplasmic reticulum chaperones ERO1A and DSBA-L involved in the assembly of adiponectin into multimeric complexes. These data show that human FGF21 does not act as an insulin sensitizer during the energy and glucose deficit of early lactation but do not rule out such a role in other physiological states.

Fibroblast growth factor-21 (FGF21) administration to early-lactating dairy cows. II. Pharmacokinetics, whole-animal performance, and lipid metabolism.

Dairy cows cope with severe energy insufficiency in early lactation by engaging in intense and sustained mobilization of fatty acids from adipose tissue. An unwanted side effect of this adaptation is excessive lipid accumulation in the liver, which in turn impairs hepatic functions. Mice experiencing increased hepatic fatty acid flux are protected from this condition through coordinated actions of the newly described hormone fibroblast growth factor-21 (FGF21) on liver and adipose tissue. The possibility of an analogous role for FGF21 in dairy cows is suggested by its rapid increase in plasma levels around parturition followed by chronically elevated levels in the first few weeks of lactation. To test this hypothesis, dairy cows were randomly assigned on d 12.6 ± 2.2 (± standard error) of lactation to receive either an excipient (control; n = 6) or recombinant human FGF21 (n = 7), first as an FGF21 bolus of 3 mg/kg of body weight (BW) followed 2 d later by a constant i.v. infusion of FGF21 at a rate of 6.3 mg/kg of metabolic BW for 9 consecutive days. After bolus administration, human FGF21 circulated with a half-life of 194 min, and its constant infusion increased total plasma concentration 117-fold over levels in excipient-infused cows. The FGF21 treatment had no effect on voluntary feed intake, milk yield, milk energy output, or net energy balance measured over the 9-d infusion or on final BW. Plasma fatty acids circulated at lower concentrations in the FGF21 group than in the control group for the 8-h period following bolus administration, but this reduction was not significant during the period of constant i.v. infusion. Treatment with FGF21 caused a 50% reduction in triglyceride content in liver biopsies taken at the end of the constant i.v. infusion without altering the mRNA abundance of key genes involved in the transport, acyl coenzyme A activation, or oxidation of fatty acids. In contrast, FGF21 treatment ablated the recovery of plasma insulin-like growth factor-1 seen in control cows during the 9-d i.v. infusion period despite a tendency for higher plasma growth hormone. This effect was associated with increased hepatic mRNA abundance of the intracellular inhibitor of growth hormone receptor trafficking, LEPROT. Overall, these data confirm the ability of FGF21 to reduce lipid accumulation in bovine liver and suggest the possibility that FGF21 does so by attenuating the hepatic influx of adipose tissue-derived fatty acids.

Variation in x-box binding protein 1 (XBP1) expression and its dependent endoplasmic reticulum chaperones does not regulate adiponectin secretion in dairy cows.

Adiponectin is an insulin-sensitizing hormone produced predominantly by adipose tissue; it circulates as oligomers of 3, 6, 18, or more units. Plasma adiponectin might be involved in the development of insulin resistance in transition dairy cows because it falls to a nadir around parturition. The possibility that this regulation occurs through a post-transcriptional mechanism was suggested in a previous study that showed unchanged adiponectin mRNA abundance combined with reduced expression of endoplasmic reticulum (ER) chaperones implicated in assembly of adiponectin oligomers. Expression of ER chaperones is controlled by x-box binding protein 1 (XBP1) and activating transcription factor 6 (ATF6), suggesting a model whereby transcriptional regulation of ER chaperones during the transition period contributes to the regulation of adiponectin production. In support of this model, XBP1 expression in adipose tissue, measured either as the active spliced XBP1 mRNA or as the total of all XBP1 mRNA isoforms, was 45% lower on d 8 of lactation than 4 wk before parturition; ATF6 mRNA abundance remained unchanged over the same period. To assess the functional importance of XBP1, preadipocytes isolated from pregnant cows were differentiated into adipocytes that secrete adiponectin. Infection of differentiating cells with an adenovirus expressing the active spliced version of bovine XBP1 did not alter adiponectin mRNA but increased the expression of ER chaperones 1.5- to 5-fold. Despite the latter, XBP1 overexpression did not affect the total amount of adiponectin secreted in medium. In additional experiments, adiponectin production was dependent on exogenous lipid in the medium and was reduced during incubation with tumor necrosis factor-α (TNFα). Accordingly, we asked whether the repressive effects of these factors on adiponectin production were related to a reduction in the expression of adiponectin or determinants of ER function (XBP1, ATF6, and ER chaperones). Exogenous lipid had no effect on the expression of any of these genes, whereas TNFα repressed adiponectin mRNA abundance by 61% but had little effect on determinants of ER function. Overall, this work shows that XBP1 is a positive regulator of ER chaperone expression in adipose tissue but provides no support for XBP1 and its dependent ER chaperones in the regulation of adiponectin production in bovine adipocytes. Mechanisms accounting for reduced plasma adiponectin in transition cows remain poorly understood.

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

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.

t10,c12-CLA decreases adiposity in peripubertal mice without dose-related detrimental effects on mammary development, inflammation status, and metabolism.

The trans 10, cis 12-conjugated linoleic acid (10,12-CLA) isomer reduces adiposity in several animal models. In the mouse, however, this effect is associated with adipose tissue inflammation, hyperinsulinemia and hepatic lipid accumulation. Moreover, 10,12-CLA was recently shown to promote mammary ductal hyperplasia and ErbB2/Her2-driven mammary cancer in the mouse. Reasons for detrimental effects of 10,12-CLA on the mouse mammary gland could relate to its effect on the mammary fat pad (MFP), which is essential for normal development. Accordingly, we hypothesized that mammary effects of 10,12-CLA were mediated through the MFP in a dose-dependent manner. Female FVB mice were fed 10,12-CLA at doses of 0%, 0.1%, 0.2%, or 0.5% of the diet from day 24 of age, and effects on mammary development and metabolism were measured on day 49. The 0.5% dose reduced ductal elongation and caused premature alveolar budding. These effects were associated with increased expression of inflammatory markers and genes shown to alter epithelial growth (IGF binding protein-5) and alveolar budding (TNF-α and receptor of activated NF-κB ligand). The 0.5% dose also caused hyperinsulinemia and hepatic lipid accumulation. In contrast, the 0.1% 10,12-CLA dose had no adverse effects on mammary development, metabolic events, and inflammatory responses, but remained effective in decreasing adipose weights and lipogenic gene expression. These results show that a low dose of 10,12-CLA reduces adiposity in the mouse without negative effects on mammary development, inflammation, and metabolism, and suggest that previously reported detrimental effects relate to the use of excessive doses.